Herbicide tolerant cotton event pDAB4468.18.07.1

ABSTRACT

Cotton event pDAB4468.18.07.1 comprises genes encoding AAD-12 and PAT, affording herbicide tolerance to cotton crops containing the event, and enabling methods for crop protection.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/589,602, filed Jan. 23, 2012, the disclosure ofwhich is hereby incorporated herein in its entirety by this reference.

BACKGROUND OF INVENTION

The gene encoding AAD-12 (aryloxyalkanoate dioxygenase-12) is capable ofimparting commercial levels of tolerance to the phenoxyacetic acidherbicides, 2,4-D and MCPA, and the pyridyloxyacetic acid herbicides,triclopyr and fluroxypyr, when expressed in transgenic plants. The geneencoding PAT (phosphinothricin acetyltransferase) is capable ofimparting tolerance to the herbicide phoshpinothricin (glufosinate) whenexpressed in transgenic plants. PAT has been successfully expressed incotton for use both as a selectable marker in producing transgeniccrops, and to impart commercial levels of tolerance to the herbicideglufosinate in transgenic plants.

The expression of transgenes in plants is known to be influenced bytheir location in the plant genome, perhaps due to chromatin structure(e.g., heterochromatin) or the proximity of transcriptional regulatoryelements (e.g., enhancers) close to the integration site (Weising etal., Ann. Rev. Genet 22:421-477, 1988). At the same time the presence ofthe transgene at different locations in the genome will influence theoverall phenotype of the plant in different ways. For this reason, it isoften necessary to screen a large number of transgenic events in orderto identify a specific transgenic event characterized by optimalexpression of an introduced gene of interest. For example, it has beenobserved in plants and in other organisms that there may be a widevariation in levels of expression of an introduced gene among events.There may also be differences in spatial or temporal patterns ofexpression, for example, differences in the relative expression of atransgene in various plant tissues, that may not correspond to thepatterns expected from transcriptional regulatory elements present inthe introduced gene construct. For this reason, it is common to producehundreds to thousands of different events and screen those events for asingle event that has desired transgene expression levels and patternsfor commercial purposes. An event that has desired levels or patterns oftransgene expression is useful for introgressing the transgene intoother genetic backgrounds by sexual outcrossing using conventionalbreeding methods. Progeny of such crosses maintain the transgeneexpression characteristics of the original transformant. This strategyis used to ensure reliable gene expression in a number of varieties thatare well adapted to local growing conditions.

It is desirable to be able to detect the presence of a particular eventin order to determine whether progeny of a sexual cross contain atransgene or group of transgenes of interest. In addition, a method fordetecting a particular event would be helpful for complying withregulations requiring the pre-market approval and labeling of food orfiber derived from recombinant crop plants, for example, or for use inenvironmental monitoring, monitoring traits in crops in the field, ormonitoring products derived from a crop harvest, as well as for use inensuring compliance of parties subject to regulatory or contractualterms.

It is possible to detect the presence of a transgenic event by anynucleic acid detection method known in the art including, but notlimited to, the polymerase chain reaction (PCR) or DNA hybridizationusing nucleic acid probes. These detection methods generally focus onfrequently used genetic elements, such as promoters, terminators, markergenes, etc., because for many DNA constructs, the coding region isinterchangeable. As a result, such methods may not be useful fordiscriminating between different events, particularly those producedusing the same DNA construct or very similar constructs unless the DNAsequence of the flanking DNA adjacent to the inserted heterologous DNAis known. For example, an event-specific PCR assay is described inUnited States Patent Application 2006/0070139 for maize eventDAS-59122-7. It would be desirable to have a simple and discriminativemethod for the identification of cotton event pDAB4468.18.07.1.

BRIEF SUMMARY OF THE INVENTION

Embodiments of the present invention relate to a new herbicide toleranttransgenic cotton transformation event, designated as cotton eventpDAB4468.18.07.1, comprising aad-12 and pat, as described herein,inserted into a specific site within the genome of a cotton cell.Representative cotton seed has been deposited with American Type CultureCollection (ATCC) ATCC Deposit No. PTA-12456. The DNA of cotton plantscontaining this event includes the junction/flanking sequences describedherein that characterize the location of the inserted DNA within thecotton genome. SEQ ID NO:1 and SEQ ID NO:2 are diagnostic for cottonevent pDAB4468.18.07.1. More particularly, sequences surrounding thejunctions at bp 2885/2886 of SEQ ID NO:1, and bp 147/148 of SEQ ID NO:2are diagnostic for cotton event pDAB4468.18.07.1. As exemplified below,sequences comprising these junctions are characteristic of DNA of cottonplants containing cotton event pDAB4468.18.07.1.

In one embodiment, the invention provides a cotton plant, or partthereof, that is tolerant to phenoxyacetic acid herbicides such as 2,4-Dand MCPA. In another embodiment, the invention provides a cotton plant,or part thereof, that is tolerant to the pyridyloxyacetic acidherbicides such as triclopyr and fluroxypyr. In an additional embodimentthe invention provides a cotton plant that has a genome comprising oneor more sequences selected from the group consisting of bp 2867-2906 ofSEQ ID NO:1; bp 2837-2936 of SEQ ID NO:1; bp 2787-2986 of SEQ ID NO:1;bp 2737-3036 of SEQ ID NO:1; bp 128-167 of SEQ ID NO:2; bp 98-197 of SEQID NO:2; bp 48-247 of SEQ ID NO:2; and bp 1-297 of SEQ ID NO:2, andcomplements thereof. In another embodiment, the invention provides seedof such plants.

In another embodiment, the invention provides a cotton plant, or partthereof, that is tolerant to compounds that are converted tophenoxyacetate auxin herbicides such as 2,4-D and MCPA (e.g., 2,4-DB,MCPB, etc.). In a further embodiment, the invention provides a cottonplant, or part thereof, that is tolerant to compounds that are convertedto pyridyloxyacetic acid herbicides such as triclopyr and fluroxypyr(e.g., triclopyrB, fluroxypyrB, etc.). The butyric acid moiety presentin the phenoxyacetate auxin and pyridyloxyacetic acid herbicides isconverted through β-oxidation to the phytotoxic form of the herbicides.The butanoic acid forms of the herbicides are themselves nonherbicidal.They are converted to their respective acid form by β-oxidation withinsusceptible plants (i.e., cotton plants), and it is the acetic acid formof the herbicide that is phytotoxic. Plants incapable of rapidβ-oxidation are not harmed by the butanoic acid herbicides. However,plants that are capable of rapid β-oxidation and can convert thebutanoic acid herbicide to the acetic form are subsequently protected byAAD-12. Accordingly, the invention provides a cotton plant that has agenome comprising one or more sequences selected from the groupconsisting of bp 2867-2906 of SEQ ID NO:1; bp 2837-2936 of SEQ ID NO:1;bp 2787-2986 of SEQ ID NO:1; bp 2737-3036 of SEQ ID NO:1; bp 128-167 ofSEQ ID NO:2; bp 98-197 of SEQ ID NO:2; bp 48-247 of SEQ ID NO:2; and bp1-297 of SEQ ID NO:2, and complements thereof. In another embodiment,the invention provides seed of such plants.

In another embodiment, the invention provides a method of controllingweeds in a cotton crop that comprises applying phenoxyacetic acidherbicides such as 2,4-D and MCPA, to the cotton crop, where the cottoncrop comprises cotton plants that have a genome containing one or moresequences selected from the group consisting of bp 2867-2906 of SEQ IDNO:1; bp 2837-2936 of SEQ ID NO:1; bp 2787-2986 of SEQ ID NO:1; bp2737-3036 of SEQ ID NO:1; bp 128-167 of SEQ ID NO:2; bp 98-197 of SEQ IDNO:2; bp 48-247 of SEQ ID NO:2; and bp 1-297 of SEQ ID NO:2, andcomplements thereof, which are diagnostic for the presence of cottonevent pDAB4468.18.07.1. In another embodiment, the invention provides amethod of controlling weeds in a cotton crop that comprises applyingpyridyloxyacetic acid herbicides, such as triclopyr and fluroxypyr, tothe cotton crop, where the cotton crop comprises cotton plants that havea genome containing one or more sequences selected from the groupconsisting of bp 2867-2906 of SEQ ID NO:1; bp 2837-2936 of SEQ ID NO:1;bp 2787-2986 of SEQ ID NO:1; bp 2737-3036 of SEQ ID NO:1; bp 128-167 ofSEQ ID NO:2; bp 98-197 of SEQ ID NO:2; bp 48-247 of SEQ ID NO:2; and bp1-297 of SEQ ID NO:2, and complements thereof, which are diagnostic forthe presence of cotton event pDAB4468.18.07.1. Presence of the aad-12gene in cotton event pDAB4468.18.07.1 imparts tolerance to phenoxyaceticacid herbicides and pyridyloxyacetic acid herbicides.

In another embodiment, the invention provides a method of controllingweeds in a cotton crop that comprises applying glufosinate herbicide tothe cotton crop, said cotton crop comprising cotton plants that have agenome containing one or more sequences selected from the groupconsisting of bp 2867-2906 of SEQ ID NO:1; bp 2837-2936 of SEQ ID NO:1;bp 2787-2986 of SEQ ID NO:1; bp 2737-3036 of SEQ ID NO:1; bp 128-167 ofSEQ ID NO:2; bp 98-197 of SEQ ID NO:2; bp 48-247 of SEQ ID NO:2; and bp1-297 of SEQ ID NO:2, and complements thereof, which are diagnostic forthe presence of cotton event pDAB4468.18.07.1. Presence of the pat genein cotton event pDAB4468.18.07.1 imparts tolerance to glufosinateherbicide.

In another embodiment, the invention provides a method of detectingcotton event pDAB4468.18.07.1 in a sample comprising cotton DNA, saidmethod comprising:

contacting said sample with a first primer at least 10 bp in length thatselectively binds to a flanking sequence within bp 1-2885 of SEQ ID NO:1or the complement thereof, and a second primer at least 10 bp in lengththat selectively binds to an insert sequence within bp 2886-3205 of SEQID NO:1 or the complement thereof; and

assaying for an amplicon generated between said primers; or

(b) contacting said sample with a first primer at least 10 bp in lengththat selectively binds to an insert sequence within bp 1-147 of SEQ IDNO:2 or the complement thereof, and a second primer at least 10 bp inlength that selectively binds to a flanking sequence within bp 148-1049of SEQ ID NO:2 or the complement thereof; and

(c) assaying for an amplicon generated between said primers.

In another embodiment, the invention provides a method of detectingcotton event pDAB4468.18.07.1 comprising:

(a) contacting said sample with a first primer that selectively binds toa flanking sequence selected from the group consisting of bp 1-2885 ofSEQ ID NO:1 and bp 148-1049 of SEQ ID NO:2, and complements thereof; anda second primer that selectively binds to SEQ ID NO:3, or the complementthereof;

(b) subjecting said sample to polymerase chain reaction; and

(c) assaying for an amplicon generated between said primers.

In another embodiment, the invention provides a method of breeding acotton plant comprising: crossing a first plant with a second cottonplant to produce a third cotton plant, said first plant comprising DNAcomprising one or more sequence selected from the group consisting of bp2867-2906 of SEQ ID NO:1; bp 2837-2936 of SEQ ID NO:1; bp 2787-2986 ofSEQ ID NO:1; bp 2737-3036 of SEQ ID NO:1; bp 128-167 of SEQ ID NO:2; bp98-197 of SEQ ID NO:2; bp 48-247 of SEQ ID NO:2; and bp 1-297 of SEQ IDNO:2, and complements thereof and assaying said third cotton plant forpresence of DNA comprising one or more sequences selected from the groupconsisting of bp 2867-2906 of SEQ ID NO:1; bp 2837-2936 of SEQ ID NO:1;bp 2787-2986 of SEQ ID NO:1; bp 2737-3036 of SEQ ID NO:1; bp 128-167 ofSEQ ID NO:2; bp 98-197 of SEQ ID NO:2; bp 48-247 of SEQ ID NO:2; and bp1-297 of SEQ ID NO:2, and complements thereof.

In another embodiment, the invention provides an isolated DNA moleculethat is diagnostic for cotton event pDAB4468.18.07.1. Such moleculesinclude, in addition to SEQ ID NOS: 1 and 2, molecules of at least 40 bpin length which comprise a polynucleotide sequence which spans the bp2885/2886 junction of SEQ ID NO:1, and molecules of at least 40 bp inlength which comprise a polynucleotide sequence which spans the bp147/148 junction of SEQ ID NO:2. Examples are bp 2867-2906 of SEQ IDNO:1; bp 2837-2936 of SEQ ID NO:1; bp 2787-2986 of SEQ ID NO:1; bp2737-3036 of SEQ ID NO:1; bp 128-167 of SEQ ID NO:2; bp 98-197 of SEQ IDNO:2; bp 48-247 of SEQ ID NO:2; and bp 1-297 of SEQ ID NO:2, andcomplements thereof.

In another embodiment, the invention provides cotton fiber, grain, seed,seed oil, or seed meal which comprises cotton event pDAB4468.18.07.1 insaid fiber, grain, seed, seed oil, or seed meal as demonstrated by saidfiber, grain, seed, seed oil, or seed meal comprising DNA comprising oneor more sequences selected from the group consisting bp 2867-2906 of SEQID NO:1; bp 2837-2936 of SEQ ID NO:1; bp 2787-2986 of SEQ ID NO:1; bp2737-3036 of SEQ ID NO:1; bp 128-167 of SEQ ID NO:2; bp 98-197 of SEQ IDNO:2; bp 48-247 of SEQ ID NO:2; and bp 1-297 of SEQ ID NO:2, andcomplements thereof.

Embodiments of the invention also include cotton plant cells and plantparts including, but not limited to, pollen, ovules, flowers, shoots,roots, and leaves, and nuclei of vegetative cells, pollen cells, seed,seed oil and seed meal, and egg cells, that contain cotton eventpDAB4468.18.07.1.

In some embodiments, cotton event pDAB4468.18.07.1 can be combined withother traits, including, for example, other herbicide tolerance gene(s)and/or insect-inhibitory proteins and transcription regulatory sequences(i.e., RNA interference, dsRNA, transcription factors, etc). Theadditional traits may be stacked into the plant genome via plantbreeding, re-transformation of the transgenic plant containing cottonevent pDAB4468.18.07.1, or addition of new traits through targetedintegration via homologous recombination.

Other embodiments include the excision of polynucleotide sequences whichcomprise cotton event pDAB4468.18.07.1, including for example, the patgene expression cassette. Upon excision of a polynucleotide sequence,the modified event may be re-targeted at a specific chromosomal sitewherein additional polynucleotide sequences are stacked with cottonevent pDAB4468.18.07.1.

In one embodiment, the present invention encompasses a cottonchromosomal target site located on chromosome 26 of the D genome betweenthe flanking sequences set forth in SEQ ID NOS:1 and 2.

In one embodiment, the present invention encompasses a method of makinga transgenic cotton plant comprising inserting a heterologous nucleicacid at a position on chromosome 26 of the D genome between the genomicsequences set forth in SEQ ID NOS:1 and 2, i.e., between bp 1-2885 ofSEQ ID NO:1 and bp 148-1049 of SEQ ID NO:2.

Additionally, embodiments of the invention also provide assays fordetecting the presence of the subject event in a sample (of cottonfibers, for example). The assays can be based on the DNA sequence of therecombinant construct, inserted into the cotton genome, and on thegenomic sequences flanking the insertion site. Kits and conditionsuseful in conducting the assays are also provided.

Embodiments of the invention also relate in part to the cloning andanalysis of the DNA sequences of the border regions resulting frominsertion of T-DNA from pDAB4468 in transgenic cotton lines. Thesesequences are unique. Based on the insert and junction sequences,event-specific primers can be and were generated. PCR analysisdemonstrated that these events can be identified by analysis of the PCRamplicons generated with these event-specific primer sets. Thus, theseand other related procedures can be used to uniquely identify cottonlines comprising the event of the subject invention.

An embodiment provides a method of controlling weeds in a cotton cropthat comprises applying phenoxyacetic acid herbicide to the cotton crop,said cotton crop comprising cotton plants comprising DNA that comprisesa sequence selected from the group consisting of bp 2867-2906 of SEQ IDNO:1; bp 2837-2936 of SEQ ID NO:1; bp 2787-2986 of SEQ ID NO:1; bp2737-3036 of SEQ ID NO:1; bp 128-167 of SEQ ID NO:2; bp 98-197 of SEQ IDNO:2; bp 48-247 of SEQ ID NO:2; and bp 1-297 of SEQ ID NO:2. In afurther aspect of this method, the phenoxyacetic acid herbicide is2,4-D. In a further aspect of this method, the phenoxyacetic acidherbicide is MCPA.

An embodiment provides a method of controlling weeds in a cotton cropthat comprises applying pyridyloxyacetic acid herbicide to the cottoncrop, said cotton crop comprising cotton plants comprising DNA thatcomprises a sequence selected from the group consisting of bp 2867-2906of SEQ ID NO:1; bp 2837-2936 of SEQ ID NO:1; bp 2787-2986 of SEQ IDNO:1; bp 2737-3036 of SEQ ID NO:1; bp 128-167 of SEQ ID NO:2; bp 98-197of SEQ ID NO:2; bp 48-247 of SEQ ID NO:2; and bp 1-297 of SEQ ID NO:2.In a further aspect of this method, the pyridyloxyacetic acid herbicideis triclopyr. In a further aspect of this method, the pyridyloxyaceticacid herbicide is fluroxypyr.

An embodiment provides a method of controlling weeds in a cotton cropthat comprises applying glufosinate herbicide to the cotton crop, saidcotton crop comprising cotton plants comprising DNA that comprises asequence selected from the group consisting of bp 2867-2906 of SEQ IDNO:1; bp 2837-2936 of SEQ ID NO:1; bp 2787-2986 of SEQ ID NO:1; bp2737-3036 of SEQ ID NO:1; bp 128-167 of SEQ ID NO:2; bp 98-197 of SEQ IDNO:2; bp 48-247 of SEQ ID NO:2; and bp 1-297 of SEQ ID NO:2.

An embodiment provides an isolated DNA sequence comprising one or moresequences selected from the group consisting of bp 2867-2906 of SEQ IDNO:1; bp 2837-2936 of SEQ ID NO:1; bp 2787-2986 of SEQ ID NO:1; bp2737-3036 of SEQ ID NO:1; bp 128-167 of SEQ ID NO:2; bp 98-197 of SEQ IDNO:2; bp 48-247 of SEQ ID NO:2; and bp 1-297 of SEQ ID NO:2.

An embodiment provides a method of breeding a cotton plant comprising:crossing a first plant with a second cotton plant to produce a thirdcotton plant, said first plant comprising DNA comprising one or moresequences selected from the group consisting of bp 2867-2906 of SEQ IDNO:1; bp 2837-2936 of SEQ ID NO:1; bp 2787-2986 of SEQ ID NO:1; bp2737-3036 of SEQ ID NO:1; bp 128-167 of SEQ ID NO:2; bp 98-197 of SEQ IDNO:2; bp 48-247 of SEQ ID NO:2; and bp 1-297 of SEQ ID NO:2, andcomplements thereof; and assaying said third cotton plant for thepresence of DNA comprising one or more sequences selected from the groupconsisting of bp 2867-2906 of SEQ ID NO:1; bp 2837-2936 of SEQ ID NO:1;bp 2787-2986 of SEQ ID NO:1; bp 2737-3036 of SEQ ID NO:1; bp 128-167 ofSEQ ID NO:2; bp 98-197 of SEQ ID NO:2; bp 48-247 of SEQ ID NO:2; and bp1-297 of SEQ ID NO:2, and complements thereof.

An embodiment provides an isolated DNA molecule comprising a junctionsequence comprising at least one sequence selected from the groupconsisting of bp 2867-2906 of SEQ ID NO:1; bp 2837-2936 of SEQ ID NO:1;bp 2787-2986 of SEQ ID NO:1; bp 2737-3036 of SEQ ID NO:1; bp 128-167 ofSEQ ID NO:2; bp 98-197 of SEQ ID NO:2; bp 48-247 of SEQ ID NO:2; and bp1-297 of SEQ ID NO:2, and complements thereof.

An embodiment provides a cotton seed comprising in its genome a DNAsequence selected from the group consisting of residues 2867-2906 of SEQID NO:1; residues 2837-2936 of SEQ ID NO:1; residues 2787-2986 of SEQ IDNO:1; residues 2737-3036 of SEQ ID NO:1; residues 128-167 of SEQ IDNO:2; residues 98-197 of SEQ ID NO:2; residues 48-247 of SEQ ID NO:2;and residues 1-297 of SEQ ID NO:2, and complements thereof. A furtherembodiment provides a cotton seed comprising in its genome AAD-12/PATcotton event pDAB4468.18.07.1 and having representative cotton seeddeposited with American Type Culture Collection under Accession No.PTA-12456. A further embodiment provides a cotton plant produced bygrowing a cotton seed of either of these two embodiments. A furtherembodiment provides a cotton seed produced by this cotton plant, whereinsaid seed comprises in its genome AAD-12/PAT cotton eventpDAB4468.18.07.1 as present in a cotton seed deposited with AmericanType Culture Collection under Accession No. PTA-12456. A furtherembodiment provides a part of this cotton plant, wherein said part isselected from the group consisting of pollen, ovule, flowers, bolls,shoots, roots, and leaves, and said part comprises AAD-12/PAT cottonevent pDAB4468.18.07.1. A further embodiment provides a compositionderived from the cotton plant or a part thereof, wherein saidcomposition is a commodity product selected from the group consisting ofcotton meal, cotton fiber, and cotton oil.

In a further embodiment, the cotton plant comprises a DNA sequencehaving at least 95% sequence identity with residues 2,886-9,253 of SEQID NO:17. An embodiment provides a progeny cotton plant of the plant ofthe above embodiment, wherein said plant exhibits tolerance tophenoxyacetic acid, pyridyloxyacetic acid, and glufosinate herbicides,and said tolerance is due to expression of a protein encoded in saidevent or said genome.

A further embodiment provides a cotton seed comprising a genomecomprising a DNA sequence having at least 95% sequence identity with SEQID NO:17. A further embodiment provides a plant produced by growing thiscotton seed.

An embodiment provides a transgenic cotton plant or part thereofcomprising cotton event pDAB4468.18.07.1, wherein representative cottonseeds comprising cotton event pDAB4468.18.07.1 have been deposited withAmerican Type Culture Collection under Accession No. PTA-12456.

SEED DEPOSIT

As part of this disclosure, at least 2500 seeds of a cotton linecomprising cotton event pDAB4468.18.07.1 have been deposited and madeavailable to the public without restriction (but subject to patentrights), with the American Type Culture Collection (ATCC), UniversityBoulevard, Manassas, Va., 20110. The deposit, designated as ATCC DepositNo. PTA-12456, was made on behalf of Dow AgroSciences LLC on Jan. 23,2012. This deposit was made and will be maintained in accordance withand under the terms of the Budapest Treaty with respect to seed depositsfor the purposes of patent procedure.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is the 5′ DNA flanking border sequence for cotton eventpDAB4468.18.07.1. Nucleotides 1-2885 are genomic sequence. Nucleotides2886-3205 are insert sequence.

SEQ ID NO:2 is the 3′ DNA flanking border sequence for cotton eventpDAB4468.18.07.1. Nucleotides 1-147 are insert sequence. Nucleotides148-1049 are genomic sequence.

SEQ ID NO:3 is the T-strand DNA sequence of pDAB4468, which is annotatedbelow in Table 1.

SEQ ID NO:4 is oligonucleotide primer 3endG1 for confirmation of 3′border genomic DNA.

SEQ ID NO:5 is oligonucleotide primer 3endG2 for confirmation of 3′border genomic DNA.

SEQ ID NO:6 is oligonucleotide primer 3endG3 for confirmation of 3′border genomic DNA.

SEQ ID NO:7 is oligonucleotide primer 5endG1 for confirmation of 5′border genomic DNA.

SEQ ID NO:8 is oligonucleotide primer 5endG2 for confirmation of 5′border genomic DNA.

SEQ ID NO:9 is oligonucleotide primer 5endG3 for confirmation of 5′border genomic DNA.

SEQ ID NO:10 is oligonucleotide primer 5endG4 for confirmation of 5′border genomic DNA.

SEQ ID NO:11 is oligonucleotide primer 5endT1 for confirmation of 5′border genomic DNA.

SEQ ID NO:12 is oligonucleotide primer 5endT2 for confirmation of 5′border genomic DNA.

SEQ ID NO:13 is oligonucleotide primer 5endT3 for confirmation of 5′border genomic DNA.

SEQ ID NO:14 is oligonucleotide primer 3endT1 for confirmation of 3′border genomic DNA.

SEQ ID NO:15 is oligonucleotide primer 3endT2 for confirmation of 3′border genomic DNA.

SEQ ID NO:16 is oligonucleotide primer 3endT3 for confirmation of 3′border genomic DNA.

SEQ ID NO:17 is the expected sequence of cotton event pDAB4468.18.07.1,including the 5′ genomic flanking sequence, pDAB4468 T-strand insert,and 3′ genomic flanking sequence.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a plasmid map of pDAB4468 containing the aad-12 and pat geneexpression cassettes.

FIG. 2 depicts the primer locations for confirming the 5′ and 3′ bordersequence of cotton event pDAB4468.18.07.1.

DETAILED DESCRIPTION OF THE INVENTION

Both ends of cotton event pDAB4468.18.07.1 insertion have been sequencedand characterized. Event specific assays were developed. The event hasalso been mapped onto the cotton genome (chromosome 26 of the D genome).The event can be introgressed into further elite lines.

As alluded to above in the Background section, the introduction andintegration of a transgene into a plant genome involves some randomevents (hence the name “event” for a given insertion that is expressed).That is, with many transformation techniques such as Agrobacteriumtransformation, the biolistic transformation (i.e., gene gun), andsilicon carbide mediated transformation (i.e., WHISKERS), it isunpredictable where in the genome a transgene will become inserted.Thus, identifying the flanking plant genomic DNA on both sides of theinsert is important for identifying a plant that has a given insertionevent. For example, PCR primers can be designed that generate a PCRamplicon across the junction region of the insert and the host genome.This PCR amplicon can be used to identify a unique or distinct type ofinsertion event.

Definitions and examples are provided herein to help describeembodiments of the present invention and to guide those of ordinaryskill in the art to practice those embodiments. Unless otherwise noted,terms are to be understood according to conventional usage by those ofordinary skill in the relevant art. The nomenclature for DNA bases asset forth at 37 CFR §1.822 is used.

As used herein, the term “progeny” denotes the offspring of anygeneration of a parent plant which comprises cotton eventpDAB4468.18.07.1.

A transgenic “event” is produced by transformation of plant cells withheterologous DNA, i.e., a nucleic acid construct that includes thetransgenes of interest, regeneration of a population of plants resultingfrom the insertion of the transgene into the genome of the plant, andselection of a particular plant characterized by insertion into aparticular genome location. The term “event” refers to the originaltransformant and progeny of the transformant that include theheterologous DNA. The term “event” also refers to progeny produced by asexual outcross between the transformant and another variety thatincludes the genomic/transgene DNA. Even after repeated back-crossing toa recurrent parent, the inserted transgene DNA and flanking genomic DNA(genomic/transgene DNA) from the transformed parent is present in theprogeny of the cross at the same chromosomal location. The term “event”also refers to DNA from the original transformant and progeny thereofcomprising the inserted DNA and flanking genomic sequence immediatelyadjacent to the inserted DNA, which would be expected to be transferredto a progeny that receives inserted DNA including the transgene ofinterest as the result of a sexual cross of one parental line thatincludes the inserted DNA (e.g., the original transformant and progenyresulting from selfing) and a parental line that does not contain theinserted DNA.

A “junction sequence” or “border sequence” spans the point at which DNAinserted into the genome is linked to DNA from the cotton native genomeflanking the insertion point, the identification or detection of one orthe other junction sequences in a plant's genetic material beingsufficient to be diagnostic for the event. Included are the DNAsequences that span the insertions in herein-described cotton events andsimilar lengths of flanking DNA. Specific examples of such diagnosticsequences are provided herein; however, other sequences that overlap thejunctions of the insertions, or the junctions of the insertions and thegenomic sequence, are also diagnostic and could be used in accordancewith embodiments of the invention.

Embodiments of the invention relate in part to event identificationusing such flanking, junction, and insert sequences. Related PCR primersand amplicons are included in embodiments of the invention. Inaccordance with embodiments of the subject invention, PCR analysismethods using amplicons that span across inserted DNA and its borderscan be used to detect or identify commercialized transgenic cottonvarieties or lines derived from the subject proprietary transgeniccotton lines.

The flanking/junction sequences are diagnostic for cotton eventpDAB4468.18.07.1. Based on these sequences, event-specific primers weregenerated. PCR analysis demonstrated that these cotton lines can beidentified in different cotton genotypes by analysis of the PCRamplicons generated with these event-specific primer sets. Thus, theseand other related procedures can be used to uniquely identify thesecotton lines. The sequences identified herein are unique.

Detection techniques of embodiments of the subject invention areespecially useful in conjunction with plant breeding, to determine whichprogeny plants comprise a given event, after a parent plant comprisingan event of interest is crossed with another plant line in an effort toimpart one or more additional traits of interest in the progeny. ThesePCR analysis methods benefit cotton breeding programs as well as qualitycontrol, especially for commercialized transgenic cotton seeds. PCRdetection kits for these transgenic cotton lines can also now be madeand used. This is also beneficial for product registration and productstewardship.

Furthermore, flanking cotton/genomic sequences can be used tospecifically identify the genomic location of each insert. Thisinformation can be used to make molecular marker systems specific toeach event. These can be used for accelerated breeding strategies and toestablish linkage data.

Still further, the flanking sequence information can be used to studyand characterize transgene integration processes, genomic integrationsite characteristics, event sorting, stability of transgenes and theirflanking sequences, and gene expression (especially related to genesilencing, transgene methylation patterns, position effects, andpotential expression-related elements such as MARS [matrix attachmentregions], and the like).

In light of all the subject disclosure, it should be clear thatembodiments of the subject invention include seeds available under theATCC Deposit No. PTA-12456. Embodiments of the invention also include aherbicide-tolerant cotton plant grown from a seed deposited with theATCC Deposit No. PTA-12456. Embodiments of the invention also includeparts of said plant, such as leaves, tissue samples, seeds produced bysaid plant, pollen, and the like (wherein these parts of the plantcomprise aad-12 and pat, and SEQ ID NOS: 1 and 2).

Still further, embodiments of the invention also include descendantand/or progeny plants of plants grown from the deposited seed,preferably a herbicide-resistant cotton plant wherein said plant has agenome comprising a detectable junction/flanking sequence as describedherein. As used herein, the term “cotton” means Gossypium hirsutum andincludes all varieties thereof that can be bred with a cotton plant.

An herbicide tolerant cotton plant of an embodiment of the invention canbe bred by first sexually crossing a first parental cotton plantconsisting of a cotton plant grown from seed of any one of the linesreferred to herein, and a second parental cotton plant, therebyproducing a plurality of first progeny plants; then selecting a firstprogeny plant that is resistant to glufosinate; selfing the firstprogeny plant, thereby producing a plurality of second progeny plants;and then selecting from the second progeny plants a plant that isresistant to glufosinate. These steps can further include theback-crossing of the first progeny plant or the second progeny plant tothe second parental cotton plant or a third parental cotton plant. Acotton crop comprising cotton seeds of an embodiment of the invention,or progeny thereof, can then be planted.

It is also to be understood that two different transgenic plants canalso be mated to produce offspring that contain two independentlysegregating, added, exogenous genes. Selfing of appropriate progeny canproduce plants that are homozygous for both added, exogenous genes.Back-crossing to a parental plant and out-crossing with a non-transgenicplant are also contemplated, as is vegetative propagation. Otherbreeding methods commonly used for different traits and crops are knownin the art. Backcross breeding has been used to transfer genes for asimply inherited, highly heritable trait into a desirable homozygouscultivar, which is the recurrent parent. The source of the trait to betransferred is called the donor parent. The resulting plant is expectedto have the attributes of the recurrent parent (e.g., cultivar) and thedesirable trait transferred from the donor parent. After the initialcross, individuals possessing the phenotype of the donor parent areselected and repeatedly crossed (backcrossed) to the recurrent parent.The resulting plant is expected to have the attributes of the recurrentparent (e.g., cultivar) and the desirable trait transferred from thedonor parent.

Likewise an herbicide tolerant cotton plant of an embodiment of theinvention can be transformed with additional transgenes using methodsknown in the art. Transformation techniques such as Agrobacteriumtransformation, the biolistic transformation (i.e., gene gun), andsilicon carbide mediated transformation (i.e., WHISKERS), can be used tointroduced additional trangene(s) into the genome of cotton eventpDAB4468.18.07.1. Selection and characterization of transgenic plantscontaining the newly inserted transgenes can be completed to identifyplants which contain a stable integrant of the novel transgene inaddition to the aad-12 and pat genes of embodiments of the invention.

The DNA molecules of embodiments of the present invention can be used asmolecular markers in a marker assisted breeding (MAB) method. DNAmolecules of embodiments of the present invention can be used in methods(such as, AFLP markers, RFLP markers, RAPD markers, SNPs, and SSRs) thatidentify genetically linked agronomically useful traits, as is known inthe art. The herbicide tolerance traits can be tracked in the progeny ofa cross with a cotton plant of embodiments of the subject invention (orprogeny thereof and any other cotton cultivar or variety) using the MABmethods. The DNA molecules are markers for this trait, and MAB methodsthat are well known in the art can be used to track the herbicidetolerance trait(s) in cotton plants where at least one cotton line ofembodiments of the subject invention, or progeny thereof, was a parentor ancestor. The methods of embodiments of the present invention can beused to identify any cotton variety having the subject event.

Methods of embodiments of the subject invention include a method ofproducing an herbicide tolerant cotton plant wherein said methodcomprises breeding with a plant of an embodiment of the subjectinvention. More specifically, said methods can comprise crossing twoplants of embodiments of the subject invention, or one plant of anembodiment of the subject invention and any other plant. Preferredmethods further comprise selecting progeny of said cross by analyzingsaid progeny for an event detectable in accordance with an embodiment ofthe subject invention and favorable varietal performance (e.g., yield).For example, embodiments of the subject invention can be used to trackthe subject event through breeding cycles with plants comprising otherdesirable traits, such as agronomic traits, disease tolerance orresistance, nematode tolerance or resistance and maturity date. Plantscomprising the subject event and the desired trait can be detected,identified, selected, and quickly used in further rounds of breeding,for example. The subject event/trait can also be combined throughbreeding, and tracked according to embodiments of the subject invention,with further insect resistant trait(s) and/or with further herbicidetolerance traits. Embodiments of the latter are plants comprising thesubject event combined with the cry1F and cry1Ac genes, which conferresistance to Pseudoplusia includens (soybean looper), Anticarsiagemmatalis (velvetbean caterpillar), Epinotia aporema, Omoidesindicatus, Rachiplusia nu, Spodoptera frugiperda, Spodoptera cosmoides,Spodoptera eridania, Heliothis virescens, Heliocoverpa zea, Spilosomavirginica and Elasmopalpus lignosellus, or with a gene encodingresistance to the herbicide dicamba.

Thus, embodiments of the subject invention can be combined with, forexample, traits encoding glyphosate resistance (e.g., resistant plant orbacterial EPSPS, GOX, GAT), glufosinate resistance (e.g., dsm-2, bar),acetolactate synthase (ALS)-inhibiting herbicide resistance (e.g.,imidazolinones [such as imazethapyr], sulfonylureas, triazolopyrimidinesulfonanilide, pyrmidinylthiobenzoates, and other chemistries [Csr1,SurA, et al.]), bromoxynil resistance (e.g., Bxn), resistance toinhibitors of HPPD (4-hydroxlphenyl-pyruvate-dioxygenase) enzyme,resistance to inhibitors of phytoene desaturase (PDS), resistance tophotosystem II inhibiting herbicides (e.g., psbA), resistance tophotosystem I inhibiting herbicides, resistance to protoporphyrinogenoxidase IX (PPO)-inhibiting herbicides (e.g., PPO-1), resistance tophenylurea herbicides (e.g., CYP76B1), dicamba-degrading enzymes (see,e.g., US 20030135879), and others could be stacked alone or in multiplecombinations to provide the ability to effectively control or preventweed shifts and/or resistance to any herbicide of the aforementionedclasses.

Additionally, cotton event pDAB4468.18.07.1 can be combined with one ormore additional input (e.g., insect resistance, pathogen resistance, orstress tolerance, et al.) or output (e.g., increased yield, improved oilprofile, improved fiber quality, et al.) traits. Thus, embodiments ofthe subject invention can be used to provide a complete agronomicpackage of improved crop quality with the ability to flexibly and costeffectively control any number of agronomic pests.

Methods to integrate a polynucleotide sequence within a specificchromosomal site of a plant cell via homologous recombination have beendescribed within the art. For instance, site specific integration asdescribed in US Patent Application Publication No. 2009/0111188 A1,herein incorporated by reference, describes the use of recombinases orintegrases to mediate the introduction of a donor polynucleotidesequence into a chromosomal target. In addition, International PatentApplication No. WO 2008/021207, herein incorporated by reference,describes zinc finger mediated-homologous recombination to integrate oneor more donor polynucleotide sequences within specific locations of thegenome. The use of recombinases such as FLP/FRT as described in U.S.Pat. No. 6,720,475, herein incorporated by reference, or CRE/LOX asdescribed in U.S. Pat. No. 5,658,772, herein incorporated by reference,can be utilized to integrate a polynucleotide sequence into a specificchromosomal site. Finally the use of meganucleases for targeting donorpolynucleotides into a specific chromosomal location was described inPuchta et al., PNAS USA 93 (1996) pp. 5055-5060.

Other methods for site specific integration within plant cells aregenerally known and applicable (Kumar et al., Trends in Plant Sci. 6(4)(2001) pp. 155-159). Furthermore, site-specific recombination systemswhich have been identified in several prokaryotic and lower eukaryoticorganisms may be applied to use in plants. Examples of such systemsinclude, but are not limited to: the R/RS recombinase system from thepSR1 plasmid of the yeast Zygosaccharomyces rouxii (Araki et al., (1985)J. Mol. Biol. 182: 191-203), and the Gin/gix system of phage Mu (Maeserand Kahlmann (1991) Mol. Gen. Genet. 230: 170-176).

In some embodiments of the present invention, it is desirable tointegrate or stack a new transgene(s) in proximity to an existingtransgenic event. The transgenic event can be considered a preferredgenomic locus which was selected based on unique characteristics such assingle insertion site, normal Mendelian segregation and stableexpression, and a superior combination of efficacy, including herbicidetolerance and agronomic performance in and across multiple environmentallocations. The newly integrated transgenes should maintain the transgeneexpression characteristics of the existing transformants. Moreover, thedevelopment of assays for the detection and confirmation of the newlyintegrated event would be overcome as the genomic flanking sequences andchromosomal location of the newly integrated event are alreadyidentified. Finally, the integration of a new transgene into a specificchromosomal location which is linked to an existing transgene wouldexpedite the introgression of the transgenes into other geneticbackgrounds by sexual out-crossing using conventional breeding methods.

In some embodiments of the present invention, it is desirable to excisepolynucleotide sequences from a transgenic event. For instance,transgene excision as described in US Patent Application Publication No.2011/0191877, herein incorporated by reference, employs zinc fingernucleases to remove a polynucleotide sequence, consisting of a geneexpression cassette, from a chromosomally integrated transgenic event.The polynucleotide sequence which is removed can be a selectable marker.Upon excision and removal of a polynucleotide sequence the modifiedtransgenic event can be retargeted by the insertion of a polynucleotidesequence. The excision of a polynucleotide sequence and subsequentretargeting of the modified transgenic event provides advantages such asre-use of a selectable marker or the ability to overcome unintendedchanges to the plant transcriptome which results from the expression ofspecific genes.

A specific site on chromosome 26 of the D genome within the cottongenome that is excellent for insertion of heterologous nucleic acids isdisclosed herein. Thus, embodiments of the subject invention providemethods to introduce heterologous nucleic acids of interest into thispre-established target site or in the vicinity of this target site.Embodiments of the subject invention also encompass a cotton seed and/ora cotton plant comprising any heterologous nucleotide sequence insertedat the disclosed target site or into the general vicinity of such site.One option to accomplish such targeted integration is to excise and/orsubstitute a different insert in place of the pat expression cassetteexemplified herein. In this regard, targeted homologous recombination,for example and without limitation, can be used in accordance withembodiments of the subject invention.

As used herein gene, event or trait “stacking” refers to the combiningof desired traits into one transgenic line. Plant breeders stacktransgenic traits by making crosses between parents that each have adesired trait and then identifying offspring that have both of thesedesired traits. Another way to stack genes is by transferring two ormore genes into the cell nucleus of a plant at the same time duringtransformation. Another way to stack genes is by re-transforming atransgenic plant with another gene of interest. For example, genestacking can be used to combine two or more different traits, includingfor example, two or more different insect traits, insect resistancetrait(s) and disease resistance trait(s), two or more herbicideresistance traits, and/or insect resistance trait(s) and herbicideresistant trait(s). The use of a selectable marker in addition to a geneof interest can also be considered gene stacking.

“Homologous recombination” refers to a reaction between any pair ofnucleotide sequences having corresponding sites containing a similarnucleotide sequence through which the two nucleotide sequences caninteract (recombine) to form a new, recombinant DNA sequence. The sitesof similar nucleotide sequence are each referred to herein as a“homology sequence.” Generally, the frequency of homologousrecombination increases as the length of the homology sequenceincreases. Thus, while homologous recombination can occur between twonucleotide sequences that are less than identical, the recombinationfrequency (or efficiency) declines as the divergence between the twosequences increases. Recombination may be accomplished using onehomology sequence on each of the donor and target molecules, therebygenerating a “single-crossover” recombination product. Alternatively,two homology sequences may be placed on each of the target and donornucleotide sequences. Recombination between two homology sequences onthe donor with two homology sequences on the target generates a“double-crossover” recombination product. If the homology sequences onthe donor molecule flank a sequence that is to be manipulated (e.g., asequence of interest), the double-crossover recombination with thetarget molecule will result in a recombination product wherein thesequence of interest replaces a DNA sequence that was originally betweenthe homology sequences on the target molecule. The exchange of DNAsequence between the target and donor through a double-crossoverrecombination event is termed “sequence replacement.”

A preferred plant, or a seed, of embodiments of the subject inventioncomprises in its genome operative aad-12 and pat nucleotide sequences,as identified herein, together with at least 20-500 or more contiguousflanking nucleotides on both sides of the insert, as identified herein.Unless indicated otherwise, reference to flanking sequences refers tothose identified with respect to SEQ ID NOS: 1 and 2. All or part ofthese flanking sequences could be expected to be transferred to progenythat receive the inserted DNA as a result of a sexual cross of aparental line that includes the event.

Embodiments of the subject invention include tissue cultures ofregenerable cells of a plant of an embodiment of the subject invention.Also included is a plant regenerated from such tissue culture,particularly where said plant is capable of expressing all themorphological and physiological properties of an exemplified variety.Preferred plants of embodiments of the subject invention have all thephysiological and morphological characteristics of a plant grown fromthe deposited seed. Embodiments of this invention further compriseprogeny of such seed and seed possessing the quality traits of interest.

As used herein, a “line” is a group of plants that display little or nogenetic variation between individuals for at least one trait. Such linesmay be created by several generations of self-pollination and selection,or vegetative propagation from a single parent using tissue or cellculture techniques.

As used herein, the terms “cultivar” and “variety” are synonymous andrefer to a line which is used for commercial production.

“Stability” or “stable” means that with respect to the given component,the component is maintained from generation to generation and,preferably, for at least three generations.

“Commercial Utility” is defined as having good plant vigor and highfertility, such that the crop can be produced by farmers usingconventional farming equipment, and the oil with the describedcomponents can be extracted from the seed using conventional crushingand extraction equipment.

“Agronomically elite” means that a line has desirable agronomiccharacteristics such as yield, maturity, disease resistance, and thelike, in addition to the herbicide tolerance due to the subjectevent(s). Any and all of these agronomic characteristics and data pointscan be used to identify such plants, either as a point or at either endor both ends of a range of characteristics used to define such plants.

As one skilled in the art will recognize in light of this disclosure,preferred embodiments of detection kits, for example, can include probesand/or primers directed to and/or comprising “junction sequences” or“transition sequences” (where the cotton genomic flanking sequence meetsthe insert sequence). For example, this includes polynucleotide probes,primers, and/or amplicons designed to identify one or both junctionsequences (where the insert meets the flanking sequence). One commondesign is to have one primer that hybridizes in the flanking region, andone primer that hybridizes in the insert. Such primers are often eachabout at least ˜15 residues in length. With this arrangement, theprimers can be used to generate/amplify a detectable amplicon thatindicates the presence of an event of an embodiment of the subjectinvention. These primers can be used to generate an amplicon that spans(and includes) a junction sequence as indicated above.

The primer(s) “touching down” in the flanking sequence is typically notdesigned to hybridize beyond about 1200 bases or so beyond the junction.Thus, typical flanking primers would be designed to comprise at least 15residues of either strand within 1200 bases into the flanking sequencesfrom the beginning of the insert. That is, primers comprising a sequenceof an appropriate size from (or hybridizing to) base pairs 2000-3205 ofSEQ ID NO:1 and/or base pairs 1-1049 of SEQ ID NO:2 are within the scopeof embodiments of the subject invention. Insert primers can likewise bedesigned anywhere on the insert, but base pairs 1-6368 of SEQ ID NO:3,can be used, for example, non-exclusively for such primer design.

One skilled in the art will also recognize that primers and probes canbe designed to hybridize, under a range of standard hybridization and/orPCR conditions wherein the primer or probe is not perfectlycomplementary to the exemplified sequence. That is, some degree ofmismatch or degeneracy can be tolerated. For an approximately 20nucleotide primer, for example, typically one or two or so nucleotidesdo not need to bind with the opposite strand if the mismatched base isinternal or on the end of the primer that is opposite the amplicon.Various appropriate hybridization conditions are provided below.Synthetic nucleotide analogs, such as inosine, can also be used inprobes. Peptide nucleic acid (PNA) probes, as well as DNA and RNAprobes, can also be used. What is important is that such probes andprimers are diagnostic for (able to uniquely identify and distinguish)the presence of an event of an embodiment of the subject invention.

It should be noted that errors in PCR amplification can occur whichmight result in minor sequencing errors, for example. That is, unlessotherwise indicated, the sequences listed herein were determined bygenerating long amplicons from cotton genomic DNAs, and then cloning andsequencing the amplicons. It is not unusual to find slight differencesand minor discrepancies in sequences generated and determined in thismanner, given the many rounds of amplification that are necessary togenerate enough amplicon for sequencing from genomic DNAs. One skilledin the art should recognize and be put on notice that any adjustmentsneeded due to these types of common sequencing errors or discrepanciesare within the scope of embodiments of the subject invention.

It should also be noted that it is not uncommon for some genomicsequence to be deleted, for example, when a sequence is inserted duringthe creation of an event. Thus, some differences can also appear betweenthe subject flanking sequences and genomic sequences listed in GENBANK,for example.

Components of the DNA sequence “insert” are illustrated in the Figuresand are discussed in more detail below in the Examples. The DNApolynucleotide sequences of these components, or fragments thereof, canbe used as DNA primers or probes in the methods of embodiments of thepresent invention.

In some embodiments of the invention, compositions and methods areprovided for detecting the presence of the transgene/genomic insertionregion, in plants and seeds and the like, from a cotton plant. DNAsequences are provided that comprise the subject 5′ transgene/genomicinsertion region junction sequence provided herein (between base pairs2885/2886 of SEQ ID NO:1), segments thereof, and complements of theexemplified sequences and any segments thereof. DNA sequences areprovided that comprise the subject 3′ transgene/genomic insertion regionjunction sequence provided herein (between base pairs 147/148 of SEQ IDNO:2), segments thereof, and complements of the exemplified sequencesand any segments thereof. The insertion region junction sequence spansthe junction between heterologous DNA inserted into the genome and theDNA from the cotton cell flanking the insertion site. Such sequences canbe diagnostic for the given event.

Based on these insert and border sequences, event-specific primers canbe generated. PCR analysis demonstrated that cotton lines of embodimentsof the subject invention can be identified in different cotton genotypesby analysis of the PCR amplicons generated with these event-specificprimer sets. These and other related procedures can be used to uniquelyidentify these cotton lines. Thus, PCR amplicons derived from suchprimer pairs are unique and can be used to identify these cotton lines.

In some embodiments, DNA sequences that comprise a contiguous fragmentof the novel transgene/genomic insertion region are an aspect of thisinvention. Included are DNA sequences that comprise a sufficient lengthof polynucleotides of transgene insert sequence and a sufficient lengthof polynucleotides of cotton genomic sequence from one or more of theaforementioned cotton plants and/or sequences that are useful as primersequences for the production of an amplicon product diagnostic for oneor more of these cotton plants.

Related embodiments pertain to DNA sequences that comprise at least 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or morecontiguous nucleotides of a transgene portion of a DNA sequenceidentified herein (such as SEQ ID NO:1 and segments thereof), orcomplements thereof, and a similar length of flanking cotton DNAsequence from these sequences, or complements thereof. Such sequencesare useful as DNA primers in DNA amplification methods. The ampliconsproduced using these primers are diagnostic for any of the cotton eventsreferred to herein. Therefore, embodiments of the invention also includethe amplicons produced by such DNA primers.

Embodiments of this invention also include methods of detecting thepresence of DNA, in a sample, that corresponds to the cotton eventreferred to herein. Such methods can comprise: (a) contacting the samplecomprising DNA with a primer set that, when used in a nucleic acidamplification reaction with DNA from at least one of these cotton event,produces an amplicon that is diagnostic for said event(s); (b)performing a nucleic acid amplification reaction, thereby producing theamplicon; and (c) detecting the amplicon.

Further detection methods of embodiments of the subject inventioninclude a method of detecting the presence of a DNA, in a sample,corresponding to said event, wherein said method comprises: (a)contacting the sample comprising DNA with a probe that hybridizes understringent hybridization conditions with DNA from said cotton event andwhich does not hybridize under the stringent hybridization conditionswith a control cotton plant (non-event-of-interest DNA); (b) subjectingthe sample and probe to stringent hybridization conditions; and (c)detecting hybridization of the probe to the DNA.

In still further embodiments, the subject invention includes methods ofproducing a cotton plant comprising cotton event pDAB4468.18.07.1 of anembodiment of the subject invention, wherein said method comprises thesteps of: (a) sexually crossing a first parental cotton line (comprisingan expression cassette of an embodiment of the present invention, whichconfers 2,4-D and glufosinate tolerance to plants of said line) and asecond parental cotton line (that lacks these herbicide tolerancetraits) thereby producing a plurality of progeny plants; and (b)selecting a progeny plant by the use of molecular markers. Such methodsmay optionally comprise the further step of back-crossing the progenyplant to the second parental cotton line to producing a true-breedingcotton plant that comprises the herbicide tolerant traits.

According to another aspect of the invention, methods of determining thezygosity of progeny of a cross with said event is provided. Said methodscan comprise contacting a sample, comprising cotton DNA, with a primerset of an embodiment of the subject invention. Said primers, when usedin a nucleic-acid amplification reaction with genomic DNA from saidcotton events, produce a first amplicon that is diagnostic for saidcotton event. Such methods further comprise performing a nucleic acidamplification reaction, thereby producing the first amplicon; detectingthe first amplicon; and contacting the sample comprising cotton DNA witha second primer set (said second primer set, when used in a nucleic-acidamplification reaction with genomic DNA from cotton plants, produces asecond amplicon comprising an endogenous sequence of the native cottongenomic DNA that does not contain the polynucleotide sequence of saidevent); and performing a nucleic acid amplification reaction, therebyproducing the second amplicon. The methods further comprise detectingthe second amplicon, and comparing the first and second amplicons in asample, wherein the presence of both amplicons indicates the zygosity ofthe transgene insertion.

DNA detection kits can be developed using the compositions disclosedherein and methods well known in the art of DNA detection. The kits areuseful for identification of the subject cotton event DNA in a sampleand can be applied to methods for breeding cotton plants containing thisDNA. The kits contain DNA sequences complementary to the amplicons, forexample, disclosed herein, or to DNA sequences complementary to DNAcontained in the transgene genetic elements of the subject events. TheseDNA sequences can be used in DNA amplification reactions or as probes ina DNA hybridization method. The kits may also contain the reagents andmaterials necessary for the performance of the detection method.

A “probe” is an isolated nucleic acid molecule to which is attached aconventional detectable label or reporter molecule (such as aradioactive isotope, ligand, chemiluminescent agent, or enzyme). Such aprobe can hybridize to a strand of a target nucleic acid, in the case ofembodiments of the present invention, to a strand of genomic DNA fromone of said cotton events, whether from a cotton plant or from a samplethat includes DNA from the event. Probes in accordance with embodimentsof the present invention include not only deoxyribonucleic orribonucleic acids but also polyamides and other probe materials thatbind specifically to a target DNA sequence and can be used to detect thepresence of that target DNA sequence.

“Primers” are isolated/synthesized nucleic acids that are annealed to atarget DNA strand by nucleic acid hybridization to form a hybrid betweenthe primer and the target DNA strand, then extended along the target DNAstrand by a polymerase, e.g., a DNA polymerase. Primer pairs ofembodiments of the present invention refer to their use foramplification of a target nucleic acid sequence, e.g., by the polymerasechain reaction (PCR) or other conventional nucleic-acid amplificationmethods.

Probes and primers are generally 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174,175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188,189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202,203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230,231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244,245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258,259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272,273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286,287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300,301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314,315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328,329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342,343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356,357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370,371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384,385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398,399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412,413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426,427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440,441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454,455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468,469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482,483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496,497, 498, 499, 500, or 1000, or 2000, or 5000 polynucleotides or more inlength. Such probes and primers hybridize specifically to a targetsequence under stringent hybridization conditions. Preferably, probesand primers in accordance with embodiments of the present invention havecomplete sequence similarity with the target sequence, although probesdiffering from the target sequence and that retain the ability tohybridize to target sequences may be designed by conventional methods.

Methods for preparing and using probes and primers are described, forexample, in Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3,ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989. PCR-primer pairs can be derived from a knownsequence, for example, by using computer programs intended for thatpurpose.

Primers and probes based on the flanking DNA and insert sequencesdisclosed herein can be used to confirm (and, if necessary, to correct)the disclosed sequences by conventional methods, e.g., by re-cloning andsequencing such sequences.

The nucleic acid probes and primers of embodiments of the presentinvention hybridize under stringent conditions to a target DNA sequence.Any conventional nucleic acid hybridization or amplification method canbe used to identify the presence of DNA from a transgenic event in asample. Nucleic acid molecules or fragments thereof are capable ofspecifically hybridizing to other nucleic acid molecules under certaincircumstances. As used herein, two nucleic acid molecules are said to becapable of specifically hybridizing to one another if the two moleculesare capable of forming an anti-parallel, double-stranded nucleic acidstructure. A nucleic acid molecule is said to be the “complement” ofanother nucleic acid molecule if they exhibit complete complementarity.As used herein, molecules are said to exhibit “complete complementarity”when every nucleotide of one of the molecules is complementary to anucleotide of the other. Molecules that exhibit complete complementaritywill generally hybridize to one another with sufficient stability topermit them to remain annealed to one another under conventional“high-stringency” conditions. Conventional high-stringency conditionsare described by Sambrook et al., 1989.

Two molecules are said to exhibit “minimal complementarity” if they canhybridize to one another with sufficient stability to permit them toremain annealed to one another under at least conventional“low-stringency” conditions. Conventional low-stringency conditions aredescribed by Sambrook et al., 1989. In order for a nucleic acid moleculeto serve as a primer or probe it need only exhibit minimalcomplementarity of sequence to be able to form a stable double-strandedstructure under the particular solvent and salt concentrations employed.

The term “stringent condition” or “stringency conditions” isfunctionally defined with regard to the hybridization of a nucleic-acidprobe to a target nucleic acid (i.e., to a particular nucleic-acidsequence of interest) by the specific hybridization procedure discussedin Sambrook et al., 1989, at 9.52-9.55. See also, Sambrook et al., 1989at 9.47-9.52 and 9.56-9.58.

Depending on the application envisioned, one can use varying conditionsof stringent conditions or polynucleotide sequence degeneracy of a probeor primer to achieve varying degrees of selectivity of hybridizationtowards the target sequence. For applications requiring highselectivity, one will typically employ relatively stringent conditionsfor hybridization of one polynucleotide sequence with a secondpolynucleotide sequence, e.g., one will select relatively low saltand/or high temperature conditions, such as provided by about 0.02 M toabout 0.15 M NaCl at temperatures of about 50° C. to about 70° C.Stringent conditions, for example, could involve washing thehybridization filter at least twice with high-stringency wash buffer(0.2×SSC, 0.1% SDS, 65° C.). Appropriate stringency conditions whichpromote DNA hybridization, for example, 6.0× sodium chloride/sodiumcitrate (SSC) at about 45 C, followed by a wash of 2.0×SSC at 50° C. areknown to those skilled in the art. For example, the salt concentrationin the wash step can be selected from a low stringency of about 2.0×SSCat 50° C. to a high stringency of about 0.2×SSC at 50° C. In addition,the temperature in the wash step can be increased from low stringencyconditions at room temperature, about 22° C., to high stringencyconditions at about 65° C. Both temperature and salt may be varied, oreither the temperature or the salt concentration may be held constantwhile the other variable is changed. Such selective conditions toleratelittle, if any, mismatch between the probe and the template or targetstrand. Detection of DNA sequences via hybridization is well-known tothose of skill in the art, and the teachings of U.S. Pat. Nos. 4,965,188and 5,176,995 are exemplary of the methods of hybridization analyses.

In a particularly preferred embodiment, a nucleic acid of an embodimentof the present invention will specifically hybridize to one or more ofthe primers (or amplicons or other sequences) exemplified or suggestedherein, including complements and fragments thereof, under highstringency conditions. In one aspect of the present invention, a markernucleic acid molecule of an embodiment of the present invention has thenucleic acid sequence as set forth herein in one of the exemplifiedsequences, or complements and/or fragments thereof.

In another aspect of the present invention, a marker nucleic acidmolecule of an embodiment of the present invention shares between 80%and 100% or 90% and 100% sequence identity with such nucleic acidsequences. In a further aspect of the present invention, a markernucleic acid molecule of an embodiment of the present invention sharesbetween 95% and 100% sequence identity with such sequence. Suchsequences may be used as markers in plant breeding methods to identifythe progeny of genetic crosses. The hybridization of the probe to thetarget DNA molecule can be detected by any number of methods known tothose skilled in the art; these can include, but are not limited to,fluorescent tags, radioactive tags, antibody based tags, andchemiluminescent tags.

Regarding the amplification of a target nucleic acid sequence (e.g., byPCR) using a particular amplification primer pair, “stringentconditions” are conditions that permit the primer pair to hybridize onlyto the target nucleic-acid sequence to which a primer having thecorresponding wild-type sequence (or its complement) would bind andpreferably to produce a unique amplification product, the amplicon.

The term “specific for (a target sequence)” indicates that a probe orprimer hybridizes under stringent hybridization conditions only to thetarget sequence in a sample comprising the target sequence.

As used herein, “amplified DNA” or “amplicon” refers to the product ofnucleic-acid amplification of a target nucleic acid sequence that ispart of a nucleic acid template. For example, to determine whether thecotton plant resulting from a sexual cross contains transgenic eventgenomic DNA from the cotton plant of an embodiment of the presentinvention, DNA extracted from a cotton plant tissue sample may besubjected to nucleic acid amplification method using a primer pair thatincludes a primer derived from flanking sequence in the genome of theplant adjacent to the insertion site of inserted heterologous DNA, and asecond primer derived from the inserted heterologous DNA to produce anamplicon that is diagnostic for the presence of the event DNA. Theamplicon is of a length and has a sequence that is also diagnostic forthe event. The amplicon may range in length from the combined length ofthe primer pairs plus one nucleotide base pair, and/or the combinedlength of the primer pairs plus about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226,227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240,241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254,255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268,269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282,283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296,297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310,311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324,325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338,339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352,353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366,367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380,381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394,395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408,409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422,423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436,437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450,451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464,465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478,479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492,493, 494, 495, 496, 497, 498, 499, or 500, 750, 1000, 1250, 1500, 1750,2000, or more nucleotide base pairs (plus or minus any of the incrementslisted above). Alternatively, a primer pair can be derived from flankingsequence on both sides of the inserted DNA so as to produce an ampliconthat includes the entire insert nucleotide sequence. A member of aprimer pair derived from the plant genomic sequence may be located adistance from the inserted DNA sequence. This distance can range fromone nucleotide base pair up to about twenty thousand nucleotide basepairs. The use of the term “amplicon” specifically excludes primerdimers that may be formed in the DNA thermal amplification reaction.

Nucleic-acid amplification can be accomplished by any of the variousnucleic-acid amplification methods known in the art, including thepolymerase chain reaction (PCR). A variety of amplification methods areknown in the art and are described, inter alia, in U.S. Pat. No.4,683,195 and U.S. Pat. No. 4,683,202. PCR amplification methods havebeen developed to amplify up to 22 kb of genomic DNA. These methods aswell as other methods known in the art of DNA amplification may be usedin the practice of embodiments of the present invention. The sequence ofthe heterologous transgene DNA insert or flanking genomic sequence froma subject cotton event can be verified (and corrected if necessary) byamplifying such sequences from the event using primers derived from thesequences provided herein followed by standard DNA sequencing of the PCRamplicon or of the cloned DNA.

The amplicon produced by these methods may be detected by a plurality oftechniques. Agarose gel electrophoresis and staining with ethidiumbromide is a common well-known method of detecting DNA amplicons.Another such method is Genetic Bit Analysis, where an DNAoligonucleotide is designed which overlaps both the adjacent flankinggenomic DNA sequence and the inserted DNA sequence. The oligonucleotideis immobilized in wells of a microwell plate. Following PCR of theregion of interest (using one primer in the inserted sequence and one inthe adjacent flanking genomic sequence), a single-stranded PCR productcan be hybridized to the immobilized oligonucleotide and serve as atemplate for a single base extension reaction using a DNA polymerase andlabeled ddNTPs specific for the expected next base. Analysis of a boundproduct can be completed via quantitating the amount of fluorescentsignal. A fluorescent signal indicates presence of the insert/flankingsequence due to successful amplification, hybridization, and single baseextension.

Another method is the Pyrosequencing technique as described by Winge(Innov. Pharma. Tech. 00:18-24, 2000). In this method an oligonucleotideis designed that overlaps the adjacent genomic DNA and insert DNAjunction. The oligonucleotide is designed to hybridize tosingle-stranded PCR product from the region of interest (one primer inthe inserted sequence and one in the flanking genomic sequence) andincubated in the presence of a DNA polymerase, ATP, sulfurylase,luciferase, apyrase, adenosine 5′ phosphosulfate and luciferin. DNTPsare added individually and the incorporation results in a light signalthat is measured. A light signal indicates the presence of the transgeneinsert/flanking sequence due to successful amplification, hybridization,and single or multi-base extension.

Fluorescence Polarization is another method that can be used to detectan amplicon of an embodiment of the present invention. Following thismethod, an oligonucleotide is designed which overlaps the genomicflanking and inserted DNA junction. The oligonucleotide is hybridized tothe single-stranded PCR product from the region of interest (one primerin the inserted DNA and one in the flanking genomic DNA sequence) andincubated in the presence of a DNA polymerase and a fluorescent-labeledddNTP. Single base extension results in incorporation of the ddNTP.Incorporation of the fluorescently labeled ddNTP can be measured as achange in polarization using a fluorometer. A change in polarizationindicates the presence of the transgene insert/flanking sequence due tosuccessful amplification, hybridization, and single base extension.

TAQMAN® (PE Applied Biosystems, Foster City, Calif.) is a method ofdetecting and quantifying the presence of a DNA sequence. Briefly, aFRET oligonucleotide probe is designed that overlaps the genomicflanking and insert DNA junction. The FRET probe and PCR primers (oneprimer in the insert DNA sequence and one in the flanking genomicsequence) are cycled in the presence of a thermostable polymerase anddNTPs. During specific amplification, the Taq DNA polymeraseproofreading mechanism releases the fluorescent moiety away from thequenching moiety on the FRET probe. A fluorescent signal indicates thepresence of the flanking/transgene insert sequence due to successfulamplification and hybridization.

Molecular Beacons have been described for use in polynucleotide sequencedetection. Briefly, a FRET oligonucleotide probe is designed thatoverlaps the flanking genomic and insert DNA junction. The uniquestructure of the FRET probe results in it containing secondary structurethat keeps the fluorescent and quenching moieties in close proximity.The FRET probe and PCR primers (one primer in the insert DNA sequenceand one in the flanking genomic sequence) are cycled in the presence ofa thermostable polymerase and dNTPs. Following successful PCRamplification, hybridization of the FRET probe to the target sequenceresults in the removal of the probe secondary structure and spatialseparation of the fluorescent and quenching moieties. A fluorescentsignal results. A fluorescent signal indicates the presence of theflanking genomic/transgene insert sequence due to successfulamplification and hybridization.

Having disclosed a location in the cotton genome that is excellent foran insertion, embodiments of the subject invention also comprise acotton seed and/or a cotton plant comprising at least one non-cottonevent pDAB4468.18.07.1 insert in the general vicinity of this genomiclocation. One option is to substitute a different insert in place of theone from cotton event pDAB4468.18.07.1 exemplified herein. In general,targeted homologous recombination, for example, is employed inparticular embodiments. This type of technology is the subject of, forexample, WO 03/080809 A2 and the corresponding published U.S.application (US 20030232410). Thus, embodiments of the subject inventioninclude plants and plant cells comprising a heterologous insert (inplace of or with multi-copies of the aad-12 or pat genes), flanked byall or a recognizable part of the flanking sequences identified herein(bp 1-2885 of SEQ ID NO:1 and bp 148-1049 of SEQ ID NO:2). An additionalcopy (or additional copies) of a aad-12 or pat gene could also betargeted for insertion in this/these manner(s).

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety to the extent they are not inconsistent with theexplicit teachings of this specification. The following examples areincluded to illustrate procedures for practicing embodiments of theinvention and to demonstrate certain preferred embodiments of theinvention. These examples should not be construed as limiting. It shouldbe appreciated by those of skill in the art that the techniquesdisclosed in the following examples represent specific approaches usedto illustrate preferred modes for its practice. However, those of skillin the art should, in light of the present disclosure, appreciate thatmany changes can be made in these specific embodiments while stillobtaining like or similar results without departing from the spirit andscope of the invention. Unless otherwise indicated, all percentages areby weight and all solvent mixture proportions are by volume unlessotherwise noted.

The following abbreviations are used unless otherwise indicated.

-   -   bp base pair    -   ° C. degrees Celsius    -   DNA deoxyribonucleic acid    -   EDTA ethylenediaminetetraacetic acid    -   kb kilobase    -   μg microgram    -   μL microliter    -   mL milliliter    -   M molar mass    -   PCR polymerase chain reaction    -   PTU plant transcription unit or expression cassette    -   SDS sodium dodecyl sulfate    -   SSC a buffer solution containing a mixture of sodium chloride        and sodium citrate, pH 7.0    -   TBE a buffer solution containing a mixture of Tris base, boric        acid and EDTA, pH 8.3

EXAMPLES Example 1: Transformation and Selection of the aad-12 and PatCotton Event pDAB4468.18.07.1

Transgenic cotton (Gossypium hirsutum) containing the cotton eventpDAB4468.18.07.1 was generated through Agrobacterium-mediatedtransformation and selected using medium containing glufosinate. Thedisarmed Agrobacterium strain EHA101 (Hood et al., 1993), carrying thebinary vector pDAB4468 (FIG. 1) containing the selectable marker, pat,and the gene of interest, aad-12, within the T-strand DNA region, wasused to initiate transformation of cotton plant variety, Coker 310. TheDNA T-strand sequence for pDAB4468 is given in SEQ ID NO:3, and isannotated below in Table 1.

TABLE 1 Gene elements located on pDAB4468. bp (SEQ ID NO: 3) Constructelement Reference   139-1,304 bp RB7 MAR v3 Thompson et al., 1997,WO9727207 1,400-2,721 bp AtUbi10 Promoter Callis, et al., (1990) J.Biol. Chem., 265: 12486-12493 2,730-3,611 bp AAD-12 WO 2007/0534823,714-4,170 bp ORF23 3′UTR U.S. Pat. No. 5,428,147 4,285-4,801 bp CsVMVPromoter Verdaguer et al., (1996) Plant Mol. Biol., 31: 1129-11394,809-5,360 bp PAT Wohlleben et al., (1988) Gene 70: 25-37 5,463-6,166bp ORF1 3′UTR Huang et al., (1990) J. Bacteriol. 172: 1814-1822

Example 2: Characterization of AAD-12 Protein from Cotton EventpDAB4468.18.07.1

The biochemical properties of the recombinant AAD-12 protein derivedfrom the transgenic cotton event pDAB4468.18.07.1 were characterized.Quantitative enzyme-linked immunosorbent assay (ELISA) was used tocharacterize the biochemical properties of the protein and confirmexpression of AAD-12 protein.

Levels of AAD-12 protein were determined in cotton eventpDAB4468.18.07.1. Samples of cotton leaf tissue were isolated from thetest plants and prepared for expression analysis. The AAD-12 protein wasextracted from cotton plant tissues with a Tris-HCl solution containingthe detergent Brij-56™ (Sigma-Aldrich, St. Louis, Mo.). The plant tissuewas centrifuged; the aqueous supernatant was collected, diluted withappropriate buffer as necessary, and analyzed using an AAD-12 ELISA kit(Beacon Diagnostics, East Falmouth, Mass.) in a sandwich format. The kitwas used following the manufacturer's suggested protocol.

Detection analysis was performed to investigate the expression stabilityand heritability both vertically (between generations) and horizontally(between lineages of the same generation) in cotton eventpDAB4468.18.07.1. The AAD-12 protein expression of cotton eventpDAB4468.18.07.1 was stable (not segregating) and consistent across alllineages.

AAD-12 protein expression levels as compared vertically (betweengenerations) were determined for greenhouse grown cotton eventpDAB4468.18.07.1 plants. Expression levels were consistent and stableacross the T₃-T₄ generations with average expression levels ofapproximately 125-140 ng/cm² of isolated AAD-12 protein.

AAD-12 protein expression levels as compared horizontally (betweenlineages of the same generation) were determined for field grown cottonevent pDAB4468.18.07.1 plants. Field expression level studies wereperformed on several cotton event pDAB4468.18.07.1 plants that comprisedthe T₄ generation. The average levels of protein expression wereconsistent and stable at approximately 50-150 ng/cm² of isolated AAD-12protein.

Example 3: Cloning and Characterization of Genomic Flanking BorderRegions of Cotton Event pDAB4468.18.07.1

Genomic flanking border regions which are adjacent to the cotton eventpDAB4468.18.07.1 T-strand insert were isolated, cloned, andcharacterized. To characterize the border regions and describe thegenomic insertion site, the genomic flanking regions of cotton eventpDAB4468.18.07.1 were isolated, comprising 2,885 bp of the 5′ genomicflanking border sequence (SEQ ID NO:1) and 902 bp of the 3′ genomicflanking border sequence (SEQ ID NO:2). The cotton eventpDAB4468.18.07.1 genomic flanking border sequences were BLASTed againstthe NCBI nucleotide database to confirm that these regions were ofcotton origin. A BLAST of the 3′ flanking border indicated that thissequence partially aligned to two different BAC Clones (Gossypiumhirsutum MX146F18 and Gossypium arboreturn GM006001). Furthermore, theBLAST search indicated that cotton event pDAB4468.18.07.1 was locatedwithin the cotton genome on chromosome 26 of the D genome. Overall, thecharacterization of the genomic flanking border sequence of cotton eventpDAB4468.18.07.1 indicated that a copy of the T-strand from pDAB4468 waspresent within the cotton genome.

Example 3.1: Confirmation of Cotton Genomic Sequences

To confirm the sequence of the genomic insertion site of cotton eventpDAB4468.18.07.1, a Polymerase Chain Reaction (PCR) was carried out withdifferent pairs of primers (FIG. 2 and Table 2 and Table 3). Genomic DNAfrom cotton event pDAB4468.18.07.1 and other transgenic ornon-transgenic cotton control lines were used as a template. The LA TaqPCR® kit was used for the reactions (TaKaRa, Japan).

TABLE 2 PCR primers and sequences used to analyze Cotton EventpDAB4468.18.07.1 SEQ ID NO: Abbreviation Sequence Name Sequence 5′ to 3′Purpose SEQ ID NO: 4 3endG1 1807_3end_genome1 TGTAGGTAAAAGATTconfirmation of 3′ AAACCATTGAGATG border genomic DNA, used with 3endT1and T3 SEQ ID NO: 5 3endG2 1807_3end_genome2 TGTGTGTTTTAGGTTconfirmation of 3′ GATTAGATTTATT border genomic DNA, used with 3endT3SEQ ID NO: 6 3endG3 1807_3end_genome3 TGAGAATAAGAGTTA confirmation of 3′TTTGAGAATAAGAGT border genomic DNA, used with 3endT3 SEQ ID NO: 7 5endG11807_5end_genome1 GAAAGAGAAATGATG confirmation of 5′ TATAAAGCAGGATTborder genomic DNA, used with 5endT1, T2, T3 SEQ ID NO: 8 5endG21807_5end_genome2 TGCTCACAGTGACTA confirmation of 5′ ACATTACATGGAAborder genomic DNA, used with 5endT1, T2, T3 SEQ ID NO: 9 5endG31807_5end_genome3 CTATTTGAGAGAAGT confirmation of 5′ AAGATGAAAGTCGborder genomic DNA, used with 5endT3 SEQ ID NO: 10 5endG41807_5end_genome4 ACAACATCGTGAATT confirmation of 5′ ACATCAATAAGAAAGborder genomic DNA, used with 5endT1 and T3 SEQ ID NO: 11 5endT1aad12_N1_primer GTGTTGCCCAGGGA Transgene Primer AGA 5′ end SEQ ID NO: 125endT2 aad12_N2_primer ATGTTGAAGCCAGGC Transgene Primer TGC 5′ end SEQID NO: 13 5endT3 ubi.for.primer CACAGAAATTTACC Transgene PrimerTTGATCACGG 5′ end SEQ ID NO: 14 3endT1 PAT.EXPRESSION CCAGAAGGTAATTATransgene Primer CASSETTE.F_primer TCCAAGATGT 3′ end SEQ ID NO: 153endT2 PAT.GOI.Primer GACAGAGCCACAAA Transgene Primer CACCACAAGA 3′ endSEQ ID NO: 16 3endT3 Orf.F.Primer AGATCGGCGGCAA Transgene PrimerTAGCTTCT 3′ end

TABLE 3 PCR conditions and reaction mixture for amplification of borderregions and event-specific sequences in cotton event pDAB4468.18.07.1.Amount (μL) of Reagent Reagent PCR Cycling Parameters LA Buffer + MgCl₂5.0 dNTP's at 2.5 mM 8.0 95° C. 5 min Primer Genome 1.0 98° C. 10 s (10μM) Primer Transgene 1.0 60° C. 30 s {close oversize brace} 35 X (10 μM)10% PVP 0.5 72° C. 4 min La Taq Polymerase 0.5 72° C. 10 min H₂O 31.5 4° C. hold DNA (20 ng/μL) 2.5 Total Volume 50.0

The 5′ genomic flanking border sequences were PCR amplified andsequenced. These reactions used aad-12 expression cassette specificprimers, (for example, 5endT1, 5endT2 and 5endT3) and primers designedaccording to the cloned 5′ end border sequence obtained from the cottongenome (for example, 5endG1 and 5endG2 and 5endG3) to amplifying agenomic DNA segment that spans the aad-12 gene and 5′ end genomicflanking border sequence. Similarly, for confirmation of the cloned 3′genomic flanking border sequence, pat expression cassette specificprimers, (for example, 3endT1, 3endT2 and 3endT3) and primers designedaccording to the cloned 3′ end border sequence (for example, 3endG1,3endG2 and 3endG3) were used to amplify a genomic DNA segment that spansthe pat gene and the 3′ end genomic flanking border sequence. DNAfragments of expected size were amplified from the genomic DNA of cottonevent pDAB4468.18.07.1 with each primer pair (one primer located on theflanking border of cotton event pDAB4468.18.07.1 and one transgenespecific primer). The control samples (other transgenic cotton lines ornon-transgenic control line, Coker 310) did not produce PCR ampliconsusing these primers. The PCR amplicons were subcloned into plasmids andsequenced. This data was used to determine the 5′ and 3′ genomicflanking border sequences located adjacent to the T-strand insert ofcotton event pDAB4468.18.07.1.

Example 4: Cotton Event pDAB4468.18.07.1 Characterization by SouthernBlot

Southern blot analysis was used to establish the integration pattern ofcotton event pDAB4468.18.07.1. These experiments generated data whichdemonstrated the integration and integrity of the aad-12, and pattransgenes within the cotton genome. Cotton event pDAB4468.18.07.1 wascharacterized as a full length, simple integration event containing asingle copy of the aad-12 and pat expression cassette from plasmidpDAB4468.

Southern blot data suggested that a T-strand fragment inserted into thegenome of cotton event pDAB4468. Detailed Southern blot analysis wasconducted using a probe specific to the aad-12 and pat genes, containedin the T-strand integration region of pDAB4468, and descriptiverestriction enzymes that have cleavage sites located within the plasmidand produce hybridizing fragments internal to the plasmid or fragmentsthat span the junction of the plasmid and cotton genomic DNA (borderfragments). The molecular weights indicated from the Southernhybridization for the combination of the restriction enzymes and theprobes were unique for the event, and established its identificationpattern. These analyses also showed that the pDAB4468 T-strand fragmenthad been inserted into the cotton genomic DNA without rearrangements ofthe aad-12 or pat expression cassettes.

Example 4.1: Cotton Leaf Sample Collection and Genomic DNA Isolation

Genomic DNA was extracted from leaf tissue harvested from individualcotton plants containing cotton event pDAB4468.18.07.1. In addition,gDNA was isolated from a conventional cotton plant, Coker 310, whichcontains the genetic background that is representative of the cottonline used for transformation and does not contain the aad-12 and patgenes. Individual genomic DNA was extracted from lyophilized leaf tissuefollowing the standard manufacturer's protocol using the Qiagen DneasyMini Prep DNA Extraction Kit® (Qiagen, CA). Following extraction, theDNA was quantified spectrofluorometrically using Pico Green® reagent(Invitrogen, Carlsbad, Calif.) and utilizing the Nanodrop®instrumentation (Invitrogen). The DNA was then visualized on an agarosegel to confirm values from the Pico Green® analysis and to determine theDNA quality.

Example 4.2: gDNA Digestion and Separation

For Southern blot characterization of cotton event pDAB4468.18.07.1, tenmicrograms (10 μg) of genomic DNA was digested. Genomic DNA from cottonevent pDAB4468.18.07.1 and the non-transgenic cotton line, Coker 310,was digested by adding approximately 10 units of selected restrictionenzyme per μg of DNA and the corresponding reaction buffer to each DNAsample. Each sample was incubated at approximately 37° C. overnight. Therestriction enzymes NsiI, NcoI, SbflI, SwaI, and NdeI were usedindividually for the digestion reactions (New England Biolabs, Ipswich,Mass.). In addition, a positive hybridization control sample wasprepared by combining plasmid DNA, pDAB4468, with genomic DNA from thenon-transgenic cotton variety, Coker310. The plasmid DNA/genomic DNAcocktail was digested using the same procedures and restriction enzymeas the test samples. After the digestions were incubated overnight, NaClwas added to a final concentration of 0.1 M to stop the restrictionenzyme digestion reaction. The digested DNA samples were precipitatedwith isopropanol. The precipitated DNA pellet was resuspended in 15 μlof 3× loading buffer (0.01% bromophenol blue, 10.0 mM EDTA, 5.0%glycerol, 1.0 mM Tris pH 7.5). The DNA samples and molecular size markerwere then electrophoresed through 0.85% agarose gels with 0.4×TAE buffer(Fisher Scientific, Pittsburgh, Pa.) at 45 volts for approximately 18-22hours to achieve fragment separation. The gels were stained withethidium bromide (Invitrogen, Carlsbad, Calif.) and the DNA wasvisualized under ultraviolet (UV) light.

Example 4.3: Southern Transfer and Membrane Treatment

Southern blot analysis was performed essentially as described byMemelink, J., Swords, K., Harry J., Hoge, C., (1994) Southern, Northern,and Western Blot Analysis. Plant Mol. Biol. Manual F1:1-23. Briefly,following electrophoretic separation and visualization of the DNAfragments, the gels were denatured with 1× Denaturation Solution (1.5 MNaOH, 20 mM EDTA) for exactly 20 minutes, and then washed with 1×Neutralization Solution (1.5 M NaPO₄, pH 7.8) for at least 20 minutes.Southern transfer was performed overnight onto nylon membranes using awicking system with 1× Transfer solution (0.25 M Sodium Pyrophosphate,pH 10). After transfer the DNA was bound to the membrane by heating at65° C. for 1 hour or by UV crosslinking followed by briefly washingmembrane with 1× Transfer solution. This process produced Southern blotmembranes ready for hybridization.

Example 4.4: DNA Probe Labeling and Hybridization

The DNA fragments bound to the nylon membrane were detected using a P³²radio-labeled probe. Probes were generated by a PCR-based method usingprimers specific to gene elements, and following standard manufacturer'sprocedure with the HotStar® Taq polymerase (Qiagen, CA). 50 ng of probeDNA specific for each gene element and 25 ng 1 Kb plus ladder(Invitrogen, Carlsbad, Calif.) were labeled with P³² using Ready to GoLabeling Beads® (Amersham, Piscataway, N.J.) following themanufacturer's instructions. Probes were purified using the NucleotideG-50® spin column from Amersham following the manufacturer's protocol.

Before addition of the P³² radiolabeled probe, the nylon membrane blotscontaining fixed DNA were blocked with Blocking buffer: (2% SDS, 0.5%BSA, 1 mM EDTA, 1 mM orthophenanthroline) for at least 2 hours at roomtemperature on a shaker, and were then pre-hybridized with 10 ml ofPerfect HYB Plus Buffer® (Sigma-Aldrich, St. Louis, Mo.) at 65° C. forat least 1 hour in a hybridization oven. Purified labeled probes weredenatured in a boiling water bath for 5 minutes and placed on ice for 5minutes and then added to the blocked and pre-hybridized membranes andplaced overnight at 65° C. in a hybridization oven.

After probe hybridization, the probe solution was discarded intoradioactive waste. The membrane blots were washed twice in theiroriginal hybridization tube with 1× Ribowash™: (200 mM Sodium Phosphate,50 mM Sodium Pyrophosphate, 10 mM EDTA, 2% SDS, pH to 7.8) wash bufferfor 15 minutes each wash in a 65° C. hybridization oven. Each wash wasdiscarded into the radioactive liquid waste following standard operatingprocedures. Membranes were removed from the tube and placed in a cleanrinsing tray and washed once more in a shaker incubator at 65° C. for anadditional 15 minutes. Membranes were wrapped in plastic wrap, placed ina film cassette and exposed to a phoshor imager screen for 1 to 3 days.Images were obtained using the BioRad Personal FX Phosphor Imager®following equipment and software guidelines.

The probes used for the hybridization are described in Table 4. The 1 KbPlus DNA Ladder (Invitrogen, Carlsbad, Calif.) was used to determinehybridizing fragment size on the Southern blots.

TABLE 4 Length of probes used in Southern analysis of cotton eventpDAB4468.18.07.1 Probe Name Genetic Element Length (bp) aad-12 aad-12671 Pat pat 525 specR Spectinomycin resistance gene 750 OriRep Ori Rep852 trfA Replication initiation protein trfA 1119

Example 4.5: Southern Blot Results

Expected and observed fragment sizes with a particular digest and probe,based on the known restriction enzyme sites of the aad-12 and pat geneexpression cassette, are given in Table 5. Expected fragment sizes arebased on the plasmid map of pDAB4468 and observed fragment sizes areapproximate results from these analyses and are based on thecorrespondence of the band with the indicated sizes of the 1 Kb Plus DNALadder.

Two types of fragments were identified from these digests andhybridizations: internal fragments where known enzyme sites flank theprobe region and are completely contained within the insertion region ofthe aad-12 expression cassette, and border fragments where a knownenzyme site is located at one end of the probe region and a second siteis expected in the cotton genome. Border fragment sizes vary by eventbecause, in most cases, DNA fragment integration sites are unique foreach event. The border fragments provide a means to locate a restrictionenzyme site relative to the integrated DNA and to evaluate the number ofDNA insertions. Southern blot analyses completed on multiple generationsof cotton lines containing cotton event pDAB4468.18.07.1 produced datawhich suggested that a low copy, intact aad-12 expression cassette fromplasmid pDAB4468 was inserted into the cotton genome thereby resultingin cotton event pDAB4468.18.07.1.

The restriction enzymes NsiI and SwaI bind and cleave unique restrictionsites within plasmid pDAB4468. Subsequently, these enzymes were selectedto characterize the aad-12 gene insert in cotton event pDAB4468.18.07.1.Border fragments of greater than 6.3 Kb or greater than 4.3 Kb werepredicted to hybridize with the probe following digests, respectively(Table 5). Single aad-12 hybridization bands of approximately 8.0 Kb andapproximately 5.0 Kb were observed when NsiI, and SwaI were used,respectively. In addition, restriction enzyme NcoI was selected tocharacterize the aad-12 gene insert. The plasmid used to produce cottonevent pDAB4468.18.07.1, pDAB4468, contained a unique NcoI restrictionsite in the expression cassette of plasmid pDAB4468, and two additionalsites in the “backbone” area of plasmid pDAB4468. A border fragment ofgreater than 3.7 Kb was predicted to hybridize with the probe followingdigests (Table 5). A single aad-12 hybridization band of approximately10.5 Kb was observed. The hybridization of the probe to bands of thissize suggests the presence of a single site of insertion for the aad-12gene in the cotton genome of cotton event pDAB4468.18.07.1. Threecombinations of restriction enzymes, Nde, NsiI+NcoI and NsiI+SbfI, wereselected to release a fragment which contains various regions of theaad-12 expression cassette and the pat expression cassette (Table 5).The NdeI restriction enzyme includes the aad-12 expression cassetteelements from the Ubi10 Promoter to the AtuORF23 UTR terminator. Apredicted fragment of 3.5 Kb was observed on blots probed with theaad-12 probe following the NdeI digest. The double digestion with NsiIand NcoI includes the aad-12 and pat expression cassette elements. Apredicted fragment of 3.5 Kb was observed on blots probed with theaad-12 probe following the double enzyme digestion. (Table 5). Thedouble digestion with NsiI and SbfI contains both aad-12 and patexpression cassette elements. A predicted fragment of 4.8 Kb wasobserved on blots probed with aad-12 following the double enzymedigestion. (Table 5).

In addition, hybridization bands were observed on blots which wereprobed with a pat probe and digested with the restriction enzymedigestions described above (NsiI, NcoI, SwaI, NdeI, NsiI+NcoI andNsiI+SbfI). The resulting blots produced fragments which indicate thatthe pat expression cassette was present in cotton eventpDAB4468.18.07.1. Table 5 lists the expected fragment sizes which arebased on the plasmid map of pDAB4468, in addition to the observedfragment sizes which resulted from the Southern blots probed with pat.These results obtained for cotton event pDAB4468.18.07.1 indicate thatan intact aad-12 expression cassette and pat expression cassette fromplasmid pDAB4468 was inserted into the cotton genome of the cotton eventpDAB4468.18.07.1.

Example 4.6: Absence of Backbone Sequences

Southern blot analysis was also conducted to verify the absence of thespectinomycin resistance gene (specR), Ori Rep element and replicationinitiation protein trfA (trf A element) in cotton eventpDAB4468.18.07.1. Following NsiI digestion and hybridization with aspecR specific probe, one expected size band of approximately 12 Kb wasobserved in the positive control sample (pDAB4468 plus Coker 310) butabsent from samples of the negative control and cotton eventpDAB4468.18.07.1. Similarly, one expected size band of approximately7.25 Kb was detected in the positive control sample (pDAB4468 plus Coker310) but absent from the samples of the negative control and cottonevent pDAB4468.18.07.1 after NcoI digestion and hybridization with amixture of OriRep specific probe and trfA specific probe. This dataindicates the absence of the spectinomycin resistance gene, Ori Repelement and replication initiation protein trfA in cotton eventpDAB4468.18.07.1.

TABLE 5 Predicted and Observed Hybridizing Fragments in Southern BlotAnalysis. Expected Fragment Observed DNA Restriction Sizes Fragment SizeProbe Enzymes Samples (bp)¹ (bp)² aad-12 NsiI pDAB4468 >11.0 Kb >11.0 KbCoker 310 None None Cotton Event  >6.3 Kb  ~8.0 Kb pDAB4468.18.07.1 NcoIpDAB4468    7.4 Kb  ~7.0 Kb Coker 310 None None Cotton Event  >3.7 Kb  10.5 Kb pDAB4468.18.07.1 SwaI pDAB4468 >11.0 Kb >11.0 Kb Coker 310None None Cotton Event  >4.3 Kb    5.0 Kb pDAB4468.18.07.1 NdeI pDAB4468   3.5 Kb    3.5 Kb Coker 310 None None Cotton Event    3.5 Kb    3.5 KbpDAB4468.18.07.1 NsiI + NcoI pDAB4468    3.5 Kb    3.5 Kb Coker 310 NoneNone Cotton Event    3.5 Kb    3.5 Kb pDAB4468.18.07.1 NsiI + SbfIpDAB4468    4.8 Kb    4.8 Kb Coker 310 None None Cotton Event    4.8 Kb   4.8 Kb pDAB4468.18.07.1 pat NsiI pDAB4468 >11.0 Kb >11.0 Kb Coker 310None None Cotton Event  >6.3 Kb  ~8.0 Kb pDAB4468.18.07.1 NcoI pDAB4468   7.4 Kb  ~7.3 Kb Coker 310 None None Cotton Event  >3.7 Kb   10.5 KbpDAB4468.18.07.1 SwaI pDAB4468 >11.0 Kb >11.0 Kb Coker 310 None NoneCotton Event >2.25 Kb    5.0 Kb pDAB4468.18.07.1 NdeI pDAB4468   5.25 Kb  5.25 Kb Coker 310 None None Cotton Event  >2.4 Kb   ~12 KbpDAB4468.18.07.1 NsiI + pDAB4468    3.5 Kb    3.5 Kb NcoI Coker 310 NoneNone Cotton Event    3.5 Kb    3.5 Kb pDAB4468.18.07.1 NsiI + pDAB4468   4.8 Kb    4.8 Kb SbfI Coker 310 None None Cotton Event    4.8 Kb   4.8 Kb pDAB4468.18.07.1 SpecR NsiI pDAB4468     12 Kb   >12 Kb Coker310 None None Cotton Event None None pDAB4468.18.07.1 OriRep NcoIpDAB4468   7.25 Kb   7.25 Kb and trfA Coker 310 None None Cotton EventNone None pDAB4468.18.07.1 ¹Expected fragment sizes are based on theplasmid map of pDAB4468. ²Observed fragment sizes are consideredapproximately from these analyses and are based on the indicated sizesof the P³²-labeled DNA Molecular Weight fragments.

Example 5: Tolerance to 2,4-D in Field Trials

The tolerance of cotton event pDAB4468.18.07.1 to postemergenceapplications of the phenoxyacetic acid herbicide, 2,4-D, was studied infield trials during the 2010 growing season. Herbicide tolerance wasassessed following a single postemergence application of 2,4-D appliedto 2-4 leaf cotton event pDAB4468.18.07.1. The application of 2,4-D oncotton plants at this stage of development represents a herbicideapplication timing typically utilized to achieve satisfactory weedcontrol by cotton growers.

2,4-D tolerance was measured by assessing cotton plants for injury at0-1 day after application (DAA), 7-8 DAA, 12-16 DAA, and 24-32 DAA.Measurements for the 0-1 day time increment were taken from 6-24 hoursafter the application. Injury was assessed by assigning a percent visualinjury rating using a linear percentage scale (1-100%) where 0%represents no visible herbicide injury and 100% represents plant death.The ratings were a composite score for any herbicide symptomologyincluding epinasty, chlorosis, leaf necrosis and plant death. Herbicidesymptomology present at 0-1 DAA was primarily epinasty and symptoms atlater evaluations were primarily chlorosis and necrosis.

Field trials were conducted at eleven locations across the United Statesin typical cotton producing areas of Alabama, Arkansas, California,Georgia, Louisiana, Mississippi, North Carolina, South Carolina, andTennessee. Trials were designed with four replications per treatment.Each replication was separated by a bare-soil alley 10-15 feet wide.Plot size for individual treatments was 2 rows by 20 feet long with rowstypically spaced 36-40 inches apart. Experimental treatments consistedof cotton event pDAB4468.18.07.1 sprayed with 2,4-D amine (Weedar 64™,NuFarm, Burr Ridge, Ill.) at either 0, 1120, 2240, or 4480 grams acidequivalent per hectare (g ae/ha) applied in 15 gallons per acre of finalspray solution. A non-transformed cotton comparator treatment was notincluded in these experiments since treatment with 2,4-D at rates muchlower than the lowest rate tested (1120 g ae/ha) are known to result inplant death.

Table 6 presents the treatment means for visual injury resulting frompostemergence application of 2,4-D. With the exception of transientepinasty 0-1 DAA, no agronomically-significant herbicide injury wasnoted in these experiments. Treatment with 2,4-D at 4480 g ae/ha (whichrepresents a rate anticipated to be >4× that required for broad spectrumweed control) resulted in little crop injury beyond the 7-8 DAAevaluation interval and demonstrates the robust tolerance of cottonevent pDAB4468.18.07.1 to 2,4-D.

TABLE 6 Percent visual injury for cotton event pDAB4468.18.07.1following postemergence application of 2,4-D. 2,4-D Rate (g ae/ha) 0-1DAA 7-8 DAA 12-16 DAA 24-32 DAA 0 0.0 0.0 0.0 0.0 1120 5.0 2.3 1.1 0.02240 11.7 4.9 2.7 0.4 4480 21.0 15.1 8.0 2.2

Example 6: Tolerance to Glufosinate in Field Trials

The tolerance of cotton event pDAB4468.18.07.1 to postemergenceapplications of glufosinate was studied in field trials during the 2010growing season. Herbicide tolerance was assessed following a singlepostemergence application of glufosinate applied to 6-8 leaf cottonevent pDAB4468.18.07.1, which represents a herbicide application timingtypically utilized with glufosinate to achieve satisfactory weedcontrol.

Glufosinate tolerance was measured by assessing cotton plants for injuryat 2-4 days after application (DAA), 7-9 DAA, 13-19 DAA and 22-29 DAA.Injury was assessed by assigning a percent visual injury rating using alinear percentage scale (1-100) where 0% represents no visible herbicideinjury and 100% represents plant death. The ratings were a compositescore for any herbicide symptomology including chlorosis, leaf necrosis,stunting and plant death. Herbicide symptomology (where present) wasprimarily chlorosis and necrosis.

Field trials were conducted at eleven locations across the United Statesin typical cotton producing areas of Alabama, Arkansas, California,Georgia, Louisiana, Mississippi, North Carolina, South Carolina, andTennessee. Trials were designed with four replications per treatment.Each replication was separated by a bare-soil alley 10-15 feet wide.Plot size for individual treatments was 2 rows by 20 feet long with rowstypically spaced 36-40 inches apart. Experimental treatments consistedof cotton event pDAB4468.18.07.1 sprayed with glufosinate ammonium(Ignite 280 SL™, Bayer, Research Triangle, N.C.) at either 0, 542, 1084,or 2168 grams acid equivalent per hectare (g ae/ha) applied in 15gallons per acre of final spray solution. A non-transformed cottoncomparator treatment was not included in these experiments sincetreatment with glufosinate at rates much lower than the lowest ratetested (542 g ae/ha) would be expected to result in plant death.

Table 7 presents the treatment means for visual injury resulting frompostemergence application of glufosinate. With the exception oftransient chlorosis in the plots treated with 2168 g ae/ha (>4× thetypical use rate) at 2-4 & 7-9 DAA, no agronomically-significantherbicide injury was noted in these experiments. Treatment withglufosinate 2168 g ae/ha (which represents a rate anticipated to be >4×that required for broad spectrum weed control) resulted in little cropinjury beyond the 7-9 DAA evaluation interval and demonstrates therobust tolerance of cotton event pDAB4468.18.07.1.

TABLE 7 Percent visual injury for cotton event pDADB4468.18.07.1following postemergence application of glufosinate. Glufosinate Rate (gae/ha) 2-4 DAA 7-9 DAA 13-19 DAA 22-29 DAA 0 0.0 0.0 0.1 0.0 542 3.1 2.30.1 0.1 1084 7.6 4.8 0.1 0.0 2168 14.8 10.8 2.2 0.3

Example 7: Tolerance to Triclopyr and Fluroxypyr in Greenhouse Trials

The tolerance of cotton event pDAB4468.18.07.1 to postemergenceapplications of the pyridyloxyacetic acid herbicides, triclopyr andfluroxypyr, was studied in greenhouse trials. Herbicide tolerance wasassessed following a single postemergence application of triclopyr orfluroxypyr.

Seeds from cotton event pDAB4468.18.07.1 were heat treated in a waterbath at 82.5° C. for 1 minute and then planted in metro mix 360 mediaand grown in a greenhouse. The temperatures in the greenhouse averaged27° C. and the photoperiod was set at 16 hours of light and eight hoursof dark, with maximum light intensity. Plants were allowed to grow untilthe 3 leaf stage of development and a non-randomized study was conductedwith three replications and three treatments for each herbicide. Plantswere sprayed in a track sprayer calibrated to deliver 187 L/ha of thecommercial formulations of triclopyr (Garton 4™, Dow AgroSciences,Indianapolis, Ind.) and fluroxypyr (Starane™, Dow AgroSciences,Indianapolis, Ind.). A non-transformed Coker 310 variety of cotton wasused as a negative control. Visual injury assessment was taken 3 DAA(days after application) and 11 DAA comparing each rate of herbicide.Injury was assessed visually as percentage of epinastic cotton-plantfoliage using a linear percentage scale (1-100%) where 0% represents novisible herbicide injury and 100% represents plant death.

Cotton event pDAB4468.18.07.1 provides tolerance to levels of fluroxypyrup to 280 g ae/ha by 3 DAA (Table 8). In addition, cotton eventpDAB4468.18.07.1 provides tolerance to triclopyr (Table 8). At 3 DAA thecotton event pDAB4468.18.07.1 plants demonstrated moderate levels oftolerance to triclopyr, but by 11 DAA the plants had recovered from theinitial epinastic response and provided robust tolerance compared to thenon-transformed control. The resulting study demonstrates that cottonevent pDAB4468.18.07.1 provides tolerance to the pyridyloxyacetic acidherbicides, triclopyr and fluroxypyr.

TABLE 8 Percent visual injury following postemergence application of theherbicides triclopyr and fluroxypyr. % Injury 3 DAA 11 DAA Rate Cottonevent Coker Cotton event Coker Herbicide (g ae/ha) pDAB4468.18.07.1Control pDAB4468.18.07.1 Control Triclopyr 140 11.8 b 33.8 a 3.0 a 36.3b Triclopyr 280 17.0 a 33.8 a 6.3 a 40.0 ab Triclopyr 560 19.3 a 36.3 a3.5 a 48.8 a Fluroxypyr 70  1.8 c 11.3 c 4.8 a 43.8 ab Fluroxypyr 140 1.0 c 27.3 b 4.3 a 36.3 b Fluroxypyr 280  5.5 c 33.3 a 8.0 a 43.8 abLeast Significant  3.98  4.78 4.07  6.95 Difference (p = .05) StandardDeviation  2.68  3.22 2.74  4.68 Means followed by same letter do notsignificantly differ (P = .05, Student -Newman-Keuls).

Example 8: Agronomic Characterization of Cotton Event pDAB4468.18.07.1

The agronomic characteristics of cotton event pDAB4468.18.07.1 werequantified in field trials conducted across geographically differentlocations throughout the cotton belt in 2010. These studies compared theagronomic performance of cotton event pDAB4468.18.07.1 (with and withoutapplications of the herbicides 2,4-D and glufosinate) to thenon-transgenic near-isoline control, Coker 310. No agronomicallymeaningful unintended differences were observed between cotton eventpDAB4468.18.07.1 and the Coker 310 control plants. The results of thesefield trials demonstrated that cotton event pDAB4468.18.07.1 wasagronomically equivalent to the Coker 310 control plants and that thepresence of the T-strand insert from pDAB4468 did not alter the expectedagronomic performance of cotton event pDAB4468.18.07.1. Additionally,the agronomic performance of cotton event pDAB4468.18.07.1 was notaltered as a result of the application of the herbicides, 2,4-D andglufosinate, as compared to the cotton event pDAB4468.18.07.1 plantswhich were not sprayed with the herbicide treatments.

Evaluations of agronomic characteristics were made for seedling vigor, %plant emergence, flower initiation, nodes after white flower (NAWF), andyield determination using the criteria indicated in Table 9. Harvestedfiber was sent to the Fiber and Biopolymer Research Institute inLubbock, Tex. for fiber evaluation using the High Volume Instrument(HVI).

TABLE 9 Agronomic characteristics evaluated in yield trials. ParameterTiming Description Scale % Plant 7 days after Cotyledons have assumed anerect Total number of emergence planting (DAP) posture and arecompletely unrolled plants that are in rows 2 and 3 of the four rowplots Seedling 7 and 28 DAP Determined by assessing the health of the1-5 scale (1 = vigor ratings plot healthy and growing well; 5 = livingbut off color, not growing, and may not survive) Days till first ~60 DAP50% of the plants in the plot have Date white flower produced at leastone white flower on a sympodial branch Nodes after 2 weeks afterRecorded the number of mainstem nodes NAWF = 5 or white flower flowerbetween the uppermost sympodial branch cutoff the (NAWF) initiation witha first position white flower and the experiment plant terminal on 5randomly selected plants per plot. The terminal node is considered to bethe uppermost with a leaf >25 mm wide. Continued the evaluation weeklyuntil the trial average reached NAWF = 5 or cutoff. Cotton yield Uponharvest Refers to the weight of pounds of cotton Pounds of cotton (lbs)per acre per acre Lint yield Upon harvest Refers to the measure of thequantity of Pounds of lint per (lbs) fiber produced on a given unit ofland. acre Presented in pounds of lint per acre

Seeds of cotton event pDAB4468.18.07.1 and seeds of the near-isolinecontrol line, Coker 310, were planted at a rate of 80 seeds per 20 ftrow with a row spacing of 34-38 inches (86.36 cm-96.52 cm). Thisplanting design was replicated four times at each site. Each site wasarranged in a complete randomized block design (CRBD) consisting of 3unsprayed blocks and 4 sprayed blocks for each replication. Plants weresprayed with the herbicides 2,4-D and glufosinate using standard fieldapplication rates and methods as described above. The center two rows(rows 2 and 3) were used to measure the agronomic characteristics andevaluate the plants. The measured agronomic characteristics wereequivalent for cotton event pDAB4468.18.07.1 as compared to the Coker310 near-isoline control plants for all of the agronomic characteristicsmeasured and statistically analyzed. One exception was identified forthe “% plant emergence” results within the herbicide sprayed plots ofcotton event pDAB4468.18.07.1. However, the “% plant emergence” datapoints were taken before the application of any herbicide and thevariability in this measurement is attributed to environmental factors.No agronomically meaningful unintended differences were observed betweencotton event pDAB4468.18.07.1 and the Coker 310 control plants. Thesefield trials demonstrated that cotton event pDAB4468.18.07.1 wasagronomically equivalent to the Coker 310 control plants and that thepresence of the T-strand insert from pDAB4468 did not alter the expectedagronomic performance of cotton event pDAB4468.18.07.1. Additionally,the agronomic performance of cotton event pDAB4468.18.07.1 was notaltered as a result of the application of the herbicides, 2,4-D andglufosinate, as compared to the cotton event pDAB4468.18.07.1 plantswhich were not sprayed with the herbicide treatments.

TABLE 10 Statistical data for treated (sprayed with herbicide) andnon-treated (unsprayed with herbicide) events as compared to annear-isoline control cotton plant. Coker Overall 310 Cotton event treat-(near- Cotton event pDAB4468.18.07.1 ment isoline pDAB4468.18.07.1sprayed with effect Parameter control) unsprayed (P-value) herbicide(P-value) (PR >F) Plant 70.3 65.8 (0.29)  64.2 (0.045) 0.553 emergence(7DAP) Vigor 1.96   2.3 (0.1362) 2.14 (0.22) 0.303 ratings Days till56.9  57.79 (0.3883) 57.79 (0.364) 0.645 first white flower (~60 DAP)NAWF 4.92  4.95 (0.8919)  5.15 (0.2539) 0.4348 (no.) Cotton 1825.432173.15 (0.4726)  1678.6 (0.6544) 0.8986 yield (lbs) Lint yield 831.63782.81 (0.2359) 792.43 (0.2887) 0.5627 (lbs)

Example 9: Expected Sequence of Cotton Event pDAB4468.18.07.1

SEQ ID NO:17 provides the expected sequence of cotton eventpDAB4468.18.07.1. This sequence contains the 5′ genomic flankingsequence, the expected T-strand insert of pDAB4468 and the 3′ genomicflanking sequence. With respect to SEQ ID NO:17, residues 1-2,885 are 5′genomic flanking sequence, residues 2,886-9,253 are residues of thepDAB4468 T-strand insert, and residues 9,254-10,156 are 3′ flankingsequence. The junction sequence or transition with respect to the 5′ endof the insert thus occurs at residues 2,885/2,886 of SEQ ID NO:17. Thejunction sequence or transition with respect to the 3′ end of the insertthus occurs at residues 9,253/9,254 of SEQ ID NO:17.

It should be noted that SEQ ID NO:17 is the expected representation ofcotton event pDAB4468.18.07.1 and was assembled from an alignment of SEQID NO:1, SEQ ID NO:2, and the T-strand of pDAB4468, SEQ ID NO:3. Theactual sequence of the T-strand insert of cotton event pDAB4468.18.07.1may slightly deviate from SEQ ID NO:17. During the transformationprocess of introducing an T-stand insert into the genome of plant cells,it is not uncommon for some deletions or other alterations of the insertto occur. Moreover, errors in PCR amplification can occur which mightresult in minor sequencing errors. For example, flanking sequenceslisted herein were determined by generating amplicons from cottongenomic DNAs, and then cloning and sequencing the amplicons. It is notunusual to find slight differences and minor discrepancies in sequencesgenerated and determined in this manner, given the many rounds ofamplification that are necessary to generate enough amplicon forsequencing from genomic DNAs. One skilled in the art should recognizeand be put on notice that any adjustments needed due to these types ofcommon sequencing errors or discrepancies are within the scope ofembodiments of the subject invention. Thus, the relevant segment of theplasmid sequence provided herein might comprise some minor variations.Thus, a plant comprising a polynucleotide having some range of identitywith the subject insert sequence is within the scope of embodiments ofthe subject invention. Specifically, a polynucleotide sequence having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the sequence of SEQ ID NO:17 is within the scope ofembodiments of the subject invention. The sequence of the flankingsequences plus insert sequence can be confirmed with reference to thedeposited seed. Thus, some differences between SEQ ID NO:17 and theactual T-strand insert of cotton event pDAB4468.18.07.1 may beidentified and are within the scope of embodiments of the presentinvention.

What is claimed is:
 1. A nucleic acid molecule comprising SEQ ID NO:17.2. A cotton seed comprising in its genome event pDAB4468.18.07.1,wherein the event comprises the polynucleotide of SEQ ID NO:17.
 3. Amethod of breeding a cotton plant comprising: crossing the cotton plantof claim 2 with a second cotton plant to produce a third cotton plant;and assaying the third cotton plant for the presence of one or morepolynucleotides selected from the group consisting of nucleotides2867-2906 of SEQ ID NO:1; nucleotides 2837-2936 of SEQ ID NO:1;nucleotides 2787-2986 of SEQ ID NO:1; nucleotides 2737-3036 of SEQ IDNO:1; nucleotides 128-167 of SEQ ID NO:2; nucleotides 98-197 of SEQ IDNO:2; nucleotides 48-247 of SEQ ID NO:2; nucleotides 1-297 of SEQ IDNO:2, and complete complements thereof.
 4. The cotton seed of claim 2,the cotton seed comprising in its genome event pDAB4468.18.07.1, whereina cotton seed representative of the seed comprising the event in itsgenome has been deposited with the American Type Culture Collectionunder Accession No. PTA-12456.
 5. A cotton plant comprising in itsgenome event pDAB4468.18.07.1, wherein the event comprises thepolynucleotide of SEQ ID NO:17.
 6. A part of the cotton plant of claim5, wherein the part is selected from the group consisting of pollen,ovule, flowers, bolls, shoots, roots, and leaves, wherein the partcomprises the polynucleotide of SEQ ID NO:17.
 7. A cotton meal or cottonoil produced from the cotton plant of claim 5, wherein the cotton mealor cotton oil comprises the polynucleotide of SEQ ID NO:17.
 8. Thecotton plant of claim 5, wherein the plant comprises in its genome eventpDAB4468.18.07.1, representative seed of the plant having been depositedwith the American Type Culture Collection under Accession No. PTA-12456.9. A method of controlling weeds in a cotton crop comprising a pluralityof the cotton plants of claim 5, the method comprising applyingphenoxyacetic acid herbicide to the cotton crop.
 10. The method of claim9, wherein the phenoxyacetic acid herbicide is 2,4-D.
 11. The method ofclaim 9, wherein the phenoxyacetic acid herbicide is MCPA.
 12. A methodof controlling weeds in a cotton crop comprising a plurality of thecotton plants of claim 5, the method comprising applyingpyridyloxyacetic acid herbicide to the cotton crop.
 13. The method ofclaim 12, wherein the pyridyloxyacetic acid herbicide is triclopyr. 14.The method of claim 12, wherein the pyridyloxyacetic acid herbicide isfluroxypyr.
 15. A method of controlling weeds in a cotton cropcomprising a plurality of the cotton plants of claim 5, the methodcomprising applying glufosinate herbicide to the cotton crop.
 16. A partof the cotton plant of claim 8, wherein the part is selected from thegroup consisting of pollen, ovule, flowers, bolls, shoots, roots, andleaves, wherein the part comprises the polynucleotide of SEQ ID NO:17.17. A cotton meal or cotton oil produced from the cotton plant of claim8, wherein the cotton meal or cotton oil comprises the polynucleotide ofSEQ ID NO:17.